Anterior endoderm cells and methods of production

ABSTRACT

Disclosed herein are cell cultures comprising PDX1-positive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified PDX1-positive endoderm cells as well as methods for enriching, isolating and purifying PDX1-positive endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of endoderm cells, such as PDX1-positive foregut endoderm cells and PDX1-negative definitive endoderm cells, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/947,999, filed on Nov. 20, 2015, which is a continuation of U.S.patent application Ser. No. 14/146,510, filed on Jan. 2, 2014, issued asU.S. Pat. No. 9,222,069, which is a continuation of U.S. patentapplication Ser. No. 11/587,735, filed on Aug. 29, 2008, issued as U.S.Pat. No. 8,647,873, which is the § 371 U.S. national stage ofInternational Application No. PCT/US2005/014239, filed Apr. 26, 2005,which was published in English under PCT Article 21(2). U.S. patentapplication Ser. No. 11/587,735 is a continuation-in-part of U.S. patentapplication Ser. No. 11/021,618, filed Dec. 23, 2004, issued as U.S.Pat. No. 7,510,876. International Application No. PCT/US2005/014239 alsoclaims the benefit of the following three provisional patentapplications: U.S. Provisional Patent Application No. 60/566,293, filedApr. 27, 2004; U.S. Provisional Patent Application No. 60/587,942, filedJul. 14, 2004; and U.S. Provisional Patent Application No. 60/586,566,filed Jul. 9, 2004.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates to compositionscomprising mammalian PDX1-positive endoderm cells and methods of making,isolating and using such cells.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells andembryonic germ (EG) cells, were first isolated in culture withoutfibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblastfeeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblottestablished continuous cultures of human ES and EG cells usingmitotically inactivated mouse feeder layers (Reubinoff et al., 2000;Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities forinvestigating early stages of human development as well as fortherapeutic intervention in several disease states, such as diabetesmellitus and Parkinson's disease. For example, the use ofinsulin-producing β-cells derived from hESCs would offer a vastimprovement over current cell therapy procedures that utilize cells fromdonor pancreases for the treatment of diabetes. However, presently it isnot known how to generate an insulin-producing β-cell from hESCs. Assuch, current cell therapy treatments for diabetes mellitus, whichutilize islet cells from donor pancreases, are limited by the scarcityof high quality islet cells needed for transplant. Cell therapy for asingle Type I diabetic patient requires a transplant of approximately8×10⁸ pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,2001a; Shapiro et al., 2001b). As such, at least two healthy donororgans are required to obtain sufficient islet cells for a successfultransplant. Human embryonic stem cells offer a source of startingmaterial from which to develop substantial quantities of high qualitydifferentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapyapplications are pluripotence and the ability to maintain these cells inculture for prolonged periods. Pluripotency is defined by the ability ofhESCs to differentiate to derivatives of all 3 primary germ layers(endoderm, mesoderm, ectoderm) which, in turn, form all somatic celltypes of the mature organism in addition to extraembryonic tissues (e.g.placenta) and germ cells. Although pluripotency imparts extraordinaryutility upon hESCs, this property also poses unique challenges for thestudy and manipulation of these cells and their derivatives. Owing tothe large variety of cell types that may arise in differentiating hESCcultures, the vast majority of cell types are produced at very lowefficiencies. Additionally, success in evaluating production of anygiven cell type depends critically on defining appropriate markers.Achieving efficient, directed differentiation is of great importance fortherapeutic application of hESCs.

In order to use hESCs as a starting material to generate cells that areuseful in cell therapy applications, it would be advantageous toovercome the foregoing problems. For example, in order to achieve thelevel of cellular material required for islet cell transplantationtherapy, it would be advantageous to efficiently direct hESCs toward thepancreatic islet/β-cell lineage at the very earliest stages ofdifferentiation.

In addition to efficient direction of the differentiation process, itwould also be beneficial to isolate and characterize intermediate celltypes along the differentiation pathway towards the pancreaticislet/β-cell lineage and to use such cells as appropriate lineageprecursors for further steps in the differentiation.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to compositions comprisingPDX1-expressing (PDX1-positive) endoderm cells as well as methods forproducing the same. Additional embodiments relate to cell populationsenriched in PDX1-positive endoderm and methods for the production ofsuch cell populations. Other embodiments relate to methods of increasingthe expression of PDX1 in endoderm cells as well as identifying factorsuseful for further differentiating PDX1-negative and/or PDX1-positiveendoderm. In some embodiments of the compositions and methods describedthroughout this application, the PDX1-positive endoderm cells arePDX1-positive foregut/midgut endoderm cells. In certain preferredembodiments of the compositions and methods described throughout thisapplication, the PDX1-positive endoderm cells are PDX1-positive foregutendoderm cells. In other preferred embodiments, the PDX1-positiveendoderm cells are PDX1-positive endoderm cells of the posterior portionof the foregut.

Some embodiments of the present invention relate to cell culturescomprising PDX1-positive foregut endoderm cells, wherein thePDX1-positive foregut endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from the anteriorportion of the gut tube. In some embodiments, the cell cultures comprisehuman cells. In such human cell cultures, PDX1-positive foregut endodermcan comprise at least about 2%, at least about 5%, at least about 10% orat least about 25% of the human cells in the culture. In someembodiments, the at least about 2%, the at least about 5%, the at leastabout 10% or the at least about 25% is calculated without respect to anyfeeder cells present in said culture. The PDX1-positive foregut endodermcells in certain embodiments of the cell cultures described herein canexpress a marker selected from the group consisting of the homeobox A13(HOXA13) gene, the homeobox C6 (HOXC6) gene and SOX17. In otherembodiments, cell cultures comprising PDX1-positive foregut endodermcells are substantially free of visceral endoderm cells, parietalendoderm cells and/or neural cells. In some embodiments, the cellcultures further comprise one of more of the following: a retinoidcompound, such as retinoic acid (RA), FGF-10 or B27.

Additional embodiments of the present invention relate to enriched,isolated or substantially purified PDX1-positive foregut endoderm cellpopulations, wherein the PDX1-positive foregut endoderm cells aremultipotent cells that can differentiate into cells, tissues or organsderived from the anterior portion of the gut tube. In some embodiments,the PDX1-positive foregut endoderm cells are derived from pluripotentcells, such as human embryonic stem cells. Other embodiments of thepresent invention, relate to a cell population which comprises cells,wherein at least about 90% of the cells are PDX1-positive foregutendoderm cells, and wherein the PDX1-positive foregut endoderm cells aremultipotent cells that can differentiate into cells, tissues or organsderived from the anterior portion of the gut tube. In preferredembodiments, the PDX-1 positive foregut endoderm cells comprise at leastabout 95% of the cells in the cell population. In even more preferredembodiments, the PDX1-positive foregut endoderm cells comprise at leastabout 98% of the cells in the cell population.

Further embodiments described herein relate to methods of producingPDX1-positive foregut endoderm cells by providing a cell culture or cellpopulation comprising definitive endoderm cells which do notsubstantially express PDX1 (PDX1-negative definitive endoderm cells)with a foregut differentiation factor, such as a retinoid. The retinoid,for example RA, can be supplied in a concentration ranging from about0.01 μM to about 50 μM. In some embodiments, the differentiation ofPDX1-negative definitive endoderm to PDX1 positive foregut endoderm isincreased by providing the cell culture or cell population with FGF-10and/or B27. FGF-10 can be supplied in a concentration ranging from about5 ng/ml to about 1000 ng/ml. In some embodiments, B27 is supplied to thecell culture or cell population at a concentration ranging from about0.1% to about 20%. FGF-10 and/or B27 can be added to the cell culture orcell population at about the same time as the retinoid or each of thefactors may be added separately with up to several hours between eachaddition. In certain embodiments, the retinoid is added to anapproximately 4-day-old PDX1-negative definitive endoderm culture. Insome embodiments, the retinoid is added to an approximately 5-day-oldPDX1-negative definitive endoderm culture.

Still other embodiments relate to methods of using a foregutdifferentiation factor to further increase the production ofPDX1-positive foregut endoderm cells in a cell culture or cellpopulation that has been contacted with a retinoid, such as RA. In suchembodiments, the differentiation of PDX1-negative definitive endoderm toPDX1 positive foregut endoderm is increased by providing the cellculture or cell population with activin A and/or activin B. Activin Aand/or activin B can be supplied in a concentration ranging from about 5ng/ml to about 1000 ng/ml. Other embodiments relate to methods ofincreasing the production of PDX1-positive foregut endoderm cells in acell culture or cell population by differentiating PDX1-negative cellsin a medium comprising a retinoid, wherein the medium has beenpreviously conditioned by the maintenance or growth of certain celltypes. Such cell types include, but are not limited to, embryonic stemcells or other pluripotent cells that have been differentiated in mediumcomprising serum or members of the TGFβ superfamily of growth factors,such as activin A, activin B, Nodal and/or bone morphogenic protein(BMP). In some embodiments, conditioned medium is supplied to the cellculture or cell population at a concentration ranging from about 1% toabout 100% of the entire growth medium. TGFβ superfamily growth factorsand/or conditioned medium can be added to the cell culture or cellpopulation at about the same time as the retinoid or each of the factorsmay be added separately with up to several hours between each addition.

Embodiments of the present invention also relate to methods of producinga cell population enriched in PDX1-positive foregut endoderm cells. Incertain embodiments, these methods comprise the step of obtaining apopulation of pluripotent cells, wherein at least one cell of thepluripotent cell population comprises at least one copy of a nucleicacid that is under the control of the PDX1 promoter. In someembodiments, the nucleic acid comprises a sequence encoding greenfluorescent protein (GFP) or a biologically active fragment thereof. Inother embodiments, additional method steps include, differentiating thepluripotent cells so as to produce PDX1-positive foregut endoderm cells,wherein the PDX1-positive foregut endoderm cells are multipotent cellsthat can differentiate into cells, tissues or organs derived from theanterior portion of the gut tube, and separating PDX1-positive cellsfrom PDX1-negative cells. In some embodiments of the methods describedherein, the differentiation step further comprises providing thepluripotent cell population with at least one growth factor of the TGFβsuperfamily in an amount sufficient to promote differentiation of thepluripotent cells to PDX1-negative definitive endoderm cells, andproviding the PDX1-negative definitive endoderm cells with a foregutdifferentiation factor in an amount sufficient to promotedifferentiation of the PDX1-negative definitive endoderm cells toPDX1-positive endoderm cells of the foregut.

Some embodiments of the present invention relate to a method ofincreasing the expression of the PDX1 gene product in SOX17-expressing(SOX17-positive) definitive endoderm cells by contacting such cells witha differentiation factor in an amount that is sufficient to increase theexpression of the PDX1 gene product. In some embodiments, thedifferentiation factor is selected from the group consisting of RA,FGF-10 and B27.

Additional embodiments of the present invention relate to a method ofidentifying a differentiation factor capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In such methods, PDX1-negativedefinitive endoderm cells are contacted with a candidate differentiationfactor and it is determined whether PDX1 expression in the cellpopulation after contact with the candidate differentiation factor hasincreased as compared to PDX1 expression in the cell population beforecontact with the candidate differentiation factor. An increase in thePDX1 expression in the cell population indicates that the candidatedifferentiation factor is capable of promoting the differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells. In some embodiments, PDX1 expression is determined byquantitative polymerase chain reaction (Q-PCR). Some embodiments of theforegoing method further comprise the step of determining expression ofthe HOXA13 and/or the HOXC6 gene in the cell population before and aftercontact with the candidate differentiation factor. In some embodiments,the candidate differentiation factor is a small molecule, for example, aretinoid, such as RA. In others, the candidate differentiation factor isa polypeptide, for example, a growth factor, such as FGF-10.

Still other embodiments of the present invention relate to a method ofidentifying a differentiation factor capable of promoting thedifferentiation of PDX1-positive foregut endoderm cells. In suchmethods, PDX1-positive foregut endoderm cells are contacted with acandidate differentiation factor and it is determined whether expressionof a marker in the population is increased or decreased after contactwith the candidate differentiation factor as compared to the expressionof the same marker in the population before contact with the candidatedifferentiation factor. An increase or decrease in the expression of themarker indicates that the candidate differentiation factor is capable ofpromoting the differentiation of PDX1-positive foregut endoderm cells.In some embodiments, marker expression is determined by Q-PCR. In someembodiments, the candidate differentiation factor is a small molecule,for example, a retinoid, such as RA. In others, the candidatedifferentiation factor is a polypeptide, for example, a growth factor,such as FGF-10.

In certain jurisdictions, there may not be any generally accepteddefinition of the term “comprising.” As used herein, the term“comprising” is intended to represent “open” language which permits theinclusion of any additional elements. With this in mind, additionalembodiments of the present inventions are described with reference tothe numbered paragraphs below:

1. A cell culture comprising human cells wherein at least about 2% ofsaid human cells are pancreatic-duodenal homoebox factor-1 (PDX1)positive foregut endoderm cells, said PDX1-positive foregut endodermcells being multipotent cells that can differentiate into cells, tissuesor organs derived from the anterior portion of the gut tube.

2. The cell culture of paragraph 1, wherein at least about 5% of saidhuman cells are PDX1-positive foregut endoderm cells.

3. The cell culture of paragraph 1, wherein at least about 10% of saidhuman cells are PDX1-positive foregut endoderm cells.

4. The cell culture of paragraph 1, wherein at least about 25% of saidhuman cells are PDX1-positive foregut endoderm cells.

5. The cell culture of paragraph 1, wherein human feeder cells arepresent in said culture, and wherein at least about 2% of human cellsother than said human feeder cells are PDX1-positive foregut endodermcells.

6. The cell culture of paragraph 1, wherein said PDX1-positive foregutendoderm cells express the homeobox A13 (HOXA13) gene.

7. The cell culture of paragraph 1, wherein said PDX1-positive foregutendoderm cells express the homeobox C6 (HOXC6) gene.

8. The cell culture of paragraph 1, wherein said PDX1-positive foregutendoderm cells express SOX17.

9. The cell culture of paragraph 1, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive foregut endoderm cells.

10. The cell culture of paragraph 1, wherein said cell culture issubstantially free of cells selected from the group consisting ofvisceral endodermal cells, parietal endodermal cells and neural cells.

11. The cell culture of paragraph 1, wherein at least about 1PDX1-positive foregut endoderm cell is present for about every 10PDX1-negative definitive endoderm cells in said cell culture.

12. The cell culture of paragraph 1, wherein at least about 1PDX1-positive foregut endoderm cell is present for about every 5PDX1-negative definitive endoderm cells in said cell culture.

13. The cell culture of paragraph 1, wherein at least about 1PDX1-positive foregut endoderm cell is present for about every 4PDX1-negative definitive endoderm cells in said cell culture.

14. The cell culture of paragraph 1 further comprising an embryonic stemcell.

15. The cell culture of paragraph 14, wherein said embryonic stem cellis derived from a tissue selected from the group consisting of themorula, the inner cell mass (ICM) of an embryo and the gonadal ridges ofan embryo.

16. The cell culture of paragraph 1 further comprising a retinoid.

17. The cell culture of paragraph 16, wherein said retinoid is retinoicacid (RA).

18. The cell culture of paragraph 1 further comprising FGF-10.

19. The cell culture of paragraph 1 further comprising B27.

20. The cell culture of paragraph 1 further comprising both RA andFGF-10.

21. The cell culture of paragraph 20 further comprising B27.

22. A cell population comprising cells wherein at least about 90% ofsaid cells are human PDX1-positive foregut endoderm cells, saidPDX1-positive foregut endoderm cells being multipotent cells that candifferentiate into cells, tissues or organs derived from the anteriorportion of the gut tube.

23. The cell population of paragraph 22, wherein at least about 95% ofsaid cells are PDX1-positive foregut endoderm cells.

24. The cell population of paragraph 22, wherein at least about 98% ofsaid cells are PDX1-positive foregut endoderm cells.

25. The cell population of paragraph 22, wherein said PDX1-positiveforegut endoderm cells express the HOXA13 gene.

26. The cell population of paragraph 22, wherein said PDX1-positiveforegut endoderm cells express the HOXC6 gene.

27. The cell population of paragraph 22, wherein said PDX1-positiveforegut endoderm cells express SOX17.

28. The cell population of paragraph 22, wherein the expression of PDX1is greater than the expression of a marker selected from the groupconsisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positiveforegut endoderm cells.

29. A method of producing PDX1-positive foregut endoderm cells, saidmethod comprising the steps of obtaining a cell population comprisingPDX1-negative definitive endoderm cells and providing said cellpopulation with a retinoid in an amount sufficient to promotedifferentiation of at least a portion of said PDX1-negative definitiveendoderm cells to PDX1-positive foregut endoderm cells, wherein saidPDX1-positive foregut endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from the anteriorportion of the gut tube.

30. The method of paragraph 29 further comprising the step of allowingsufficient time for PDX1-positive foregut endoderm cells to form,wherein said sufficient time for PDX1-positive foregut endoderm cells toform has been determined by detecting the presence of PDX1-positiveforegut endoderm cells in said cell population.

31. The method of paragraph 29, wherein at least about 2% of saidPDX1-negative definitive endoderm cells differentiate into PDX1-positiveforegut endoderm cells.

32. The method of paragraph 29, wherein at least about 5% of saidPDX1-negative definitive endoderm cells differentiate into PDX1-positiveforegut endoderm cells.

33. The method of paragraph 29, wherein at least about 10% of saidPDX1-negative definitive endoderm cells differentiate into PDX1-positiveforegut endoderm cells.

34. The method of paragraph 29, wherein at least about 25% of saidPDX1-negative definitive endoderm cells differentiate into PDX1-positiveforegut endoderm cells.

35. The method of paragraph 29, wherein detecting the presence ofPDX1-positive foregut endoderm cells in said cell population comprisesdetecting the expression of PDX1.

36. The method of paragraph 35, wherein the expression of PDX1 isgreater than the expression of a marker selected from the groupconsisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in saidPDX1-positive foregut endoderm cells.

37. The method of paragraph 35, wherein the expression of said PDX1 isdetermined by quantitative polymerase chain reaction (Q-PCR).

38. The method of paragraph 35, wherein the expression of PDX1 isdetermined by immunocytochemistry.

39. The method of paragraph 29, wherein said retinoid is RA.

40. The method of paragraph 39, wherein RA is provided in aconcentration ranging from about 0.01 μM to about 50 μM.

41. The method of paragraph 39, wherein RA is provided in aconcentration ranging from about 0.04 μM to about 20 μM.

42. The method of paragraph 39, wherein RA is provided in aconcentration ranging from about 0.1 μM to about 10 μM.

43. The method of paragraph 39, wherein RA is provided in aconcentration ranging from about 0.2 μM to about 2.5 μM.

44. The method of paragraph 39, wherein RA is provided in aconcentration ranging from about 0.5 μM to about 1.5 μM.

45. The method of paragraph 39, wherein RA is provided in aconcentration of about 1 μM.

46. The method of paragraph 39, wherein RA is provided when said cultureis about 4-days-old.

47. The method of paragraph 29 further comprising providing to saidculture a factor selected from the group consisting of FGF-10, FGF-4,activin A, activin B, B27, conditioned medium, and combinations of saidfactors in an amount sufficient to enhance the production ofPDX1-positive foregut endoderm cells.

48. The method of paragraph 47, wherein said factor is selected from thegroup consisting of FGF-10, FGF-4, activin A and activin B.

49. The method of paragraph 48, wherein said factor is provided in aconcentration ranging from about 10 ng/ml to about 500 ng/ml.

50. The method of paragraph 48, wherein said factor is provided in aconcentration ranging from about 20 ng/ml to about 200 ng/ml.

51. The method of paragraph 48, wherein said factor is provided in aconcentration ranging from about 25 ng/ml to about 75 ng/ml.

52. The method of paragraph 48, wherein said factor is provided in aconcentration about 50 ng/ml.

53. The method of paragraph 48, wherein said factor is provided atapproximately the same time as said retinoid.

54. The method of paragraph 47 wherein said factor is B27.

55. The method of paragraph 54, wherein B27 is provided in aconcentration ranging from about 0.1% to about 20% of the total medium.

56. The method of paragraph 54, wherein B27 is provided in aconcentration ranging from about 0.2% to about 5% of the total medium.

57. The method of paragraph 54, wherein B27 is provided in aconcentration ranging from 0.5% to about 2% of the total medium.

58. The method of paragraph 54, wherein B27 is provided in aconcentration of about 1% of the total medium.

59. The method of paragraph 54, wherein B27 is provided at approximatelythe same time as said retinoid.

60. The method of paragraph 47 wherein said factor is conditionedmedium.

61. The method of paragraph 60, wherein conditioned medium is providedin a concentration ranging from about 10% to about 100% of the totalmedium.

62. The method of paragraph 60, wherein conditioned medium is providedin a concentration ranging from about 20% to about 80% of the totalmedium.

63. The method of paragraph 60, wherein conditioned medium is providedin a concentration ranging from about 40% to about 60% of the totalmedium.

64. The method of paragraph 60, wherein conditioned medium is providedin a concentration of about 50% of the total medium.

65. The method of paragraph 60, wherein conditioned medium is providedat approximately the same time as said retinoid.

66. The method of paragraph 60, wherein conditioned medium is preparedby contacting differentiated human embryonic stem cells (hESCs) with acell culture medium for about 24 hours.

67. The method of paragraph 66, wherein said hESCs are differentiatedfor about 5 days in a cell culture medium selected from the groupconsisting of RPMI supplemented with 3% serum, low serum RPMIsupplemented with activin A and low serum RPMI supplemented with BMP4.

68. A PDX1-positive foregut endoderm cell produced by the method ofparagraph 29.

69. A method of producing a cell population enriched in PDX1-positiveforegut endoderm cells, said method comprising the steps of obtaining apopulation of pluripotent cells, wherein at least one cell of saidpluripotent cell population comprises at least one copy of a nucleicacid under the control of the PDX1 promoter, said nucleic acidcomprising a sequence encoding green fluorescent protein (GFP) or abiologically active fragment thereof, differentiating said pluripotentcells so as to produce PDX1-positive foregut endoderm cells, saidPDX1-positive foregut endoderm cells being multipotent cells that candifferentiate into cells, tissues or organs derived from the anteriorportion of the gut tube and separating said PDX1-positive foregutendoderm cells from PDX1-negative cells.

70. The method of paragraph 69, wherein said enriched cell populationcomprises at least about 95% PDX1-positive foregut endoderm cells.

71. The method of paragraph 69, wherein said enriched cell populationcomprises at least about 98% PDX1-positive foregut endoderm cells.

72. The method of paragraph 69, wherein the differentiating step furthercomprises, providing said pluripotent cell population with at least onegrowth factor of the TGFβ superfamily in an amount sufficient to promotedifferentiation of said pluripotent cells to PDX1-negative definitiveendoderm cells, and providing said PDX1-negative definitive endodermcells with a retinoid in an amount sufficient to promote differentiationof said PDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells.

73. The method of paragraph 72, wherein said retinoid is RA.

74. An enriched population of PDX1-positive foregut endoderm cellsproduced by the method of paragraph 69.

75. A method of increasing the expression of the PDX1 gene product in aSOX17 expressing definitive endoderm cell, said method comprisingcontacting said definitive endoderm cell with a differentiation factorin an amount sufficient to increase expression of the PDX1 gene product.

76. The method of paragraph 75, wherein said differentiation factor is aretinoid.

77. The method of paragraph 76, wherein said differentiation factor isRA.

78. The method of paragraph 75, wherein said differentiation factor isselected from the group consisting of FGF-10, FGF-4, activin A, activinB, B27, conditioned medium and combinations of said factors.

79. A method of identifying a differentiation factor capable ofpromoting the differentiation of PDX1-negative definitive endoderm cellsto PDX1-positive foregut endoderm cells, said method comprising thesteps of obtaining a population comprising PDX1-negative definitiveendoderm cells, contacting said population comprising PDX1-negativedefinitive endoderm cells with a candidate differentiation factor anddetermining if PDX1 expression in said cell population after contactwith said candidate differentiation factor has increased as compared toPDX1 expression in said cell population before contact with saidcandidate differentiation factor, wherein an increase in said PDX1expression in said cell population indicates that said candidatedifferentiation factor is capable of promoting the differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells, said PDX1-positive foregut endoderm cells beingmultipotent cells that can differentiate into cells, tissues or organsderived from the anterior portion of the gut tube.

80. The method of paragraph 79, wherein said PDX1 expression isdetermined by Q-PCR.

81. The method of paragraph 79 further comprising the step ofdetermining the expression of the HOXA13 gene in said cell populationbefore and after contact with said candidate differentiation factor.

82. The method of paragraph 79 further comprising the step ofdetermining the expression of the HOXC6 gene in said cell populationbefore and after contact with said candidate differentiation factor.

83. The method of paragraph 79, wherein said candidate differentiationfactor is a small molecule.

84. The method of paragraph 83, wherein said small molecule is aretinoid.

85. The method of paragraph 84, wherein said retinoid is RA.

86. The method of paragraph 79, wherein said candidate differentiationfactor is a polypeptide.

87. The method of paragraph 79, wherein said candidate differentiationfactor is a growth factor.

88. The method of paragraph 79, wherein said candidate differentiationfactor is FGF-10.

89. A method of identifying a differentiation factor capable ofpromoting the differentiation of PDX1-positive foregut endoderm cells,said method comprising the steps of obtaining a population comprisingPDX1-positive foregut endoderm cells, contacting said populationcomprising PDX1-positive foregut endoderm cells with a candidatedifferentiation factor and determining if expression of a marker in saidpopulation is increased or decreased after contact with said candidatedifferentiation factor, as compared to expression of the same marker insaid population before contact with said candidate differentiationfactor, wherein an increase or decrease in expression of said marker insaid population indicates that said candidate differentiation factor iscapable of promoting the differentiation of PDX1-positive foregutendoderm cells.

90. The method of paragraph 81, wherein said marker expression isdetermined by Q-PCR.

91. The method of paragraph 81, wherein said candidate differentiationfactor is a small molecule.

92. The method of paragraph 81, wherein said candidate differentiationfactor is a polypeptide.

93. The method of paragraph 81, wherein said candidate differentiationfactor is a growth factor.

94. A vector comprising a reporter gene operably linked to a PDX1control region.

95. The vector of paragraph 94, wherein said reporter gene is EGFP.

96. A cell comprising the vector of paragraph 94.

97. A cell comprising a reporter gene operably linked to a PDX1 controlregion.

98. The cell of paragraph 97, wherein said reporter gene operably linkedto said PDX1 control region is integrated into a chromosome.

99. The cell of paragraph 97, wherein said reporter gene is EGFP.

100. The cell of paragraph 97, wherein said cell is pluripotent.

101. The cell of paragraph 100, wherein said cell is a hESC.

102. The cell of paragraph 97, wherein said cell is a definitiveendoderm cell.

103. The cell of paragraph 97, wherein said cell is a PDX1-positiveforegut endoderm cell.

104. A conditioned medium prepared by the steps of contacting fresh cellculture medium with a population of differentiated hESCs for about 24hours, wherein said hESCs have been differentiated for about 5 days in acell culture medium selected from the group consisting of RPMIsupplemented with 3% serum, low serum RPMI supplemented with activin Aand low serum RPMI supplemented with BMP4 and removing said populationof differentiated hESCs from the medium.

105. The conditioned medium of paragraph 104, wherein said fresh cellculture medium is RPMI.

106. The conditioned medium of paragraph 105, wherein said RPMI is lowserum RPMI.

107. A method for conditioning medium, said method comprising the stepsof contacting fresh cell culture medium with a population ofdifferentiated hESCs for about 24 hours, wherein said hESCs have beendifferentiated for about 5 days in a cell culture medium selected fromthe group consisting of RPMI supplemented with 3% serum, low serum RPMIsupplemented with activin A and low serum RPMI supplemented with BMP4and removing said population of differentiated hESCs from the medium.

108. The method of paragraph 107, wherein said fresh cell culture mediumis RPMI.

109. The method of paragraph 108, wherein said RPMI is low serum RPMI.

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.

Additional embodiments of the present invention may also be found inU.S. Provisional Patent Application No. 60/532,004, entitled DEFINITIVEENDODERM, filed Dec. 23, 2003; U.S. Provisional Patent Application No.60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S.Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,2004; U.S. Provisional Patent Application No. 60/587,942, entitledCHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM, filed Jul. 14, 2004; and U.S. patent application Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosures of which are incorporated herein by reference in theirentireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a proposed differentiation pathway for theproduction of beta-cells from hESCs. The first step in the pathwaycommits the ES cell to the definitive endoderm lineage and alsorepresents the first step prior to further differentiation events topancreatic endoderm, endocrine endoderm, or islet/beta-cells. The secondstep in the pathway shows the conversion of SOX17-positive/PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm. Some factorsuseful for mediating these transitions are italicized. Relevant markersfor defining the target cells are underlined.

FIG. 2 is a diagram of the human SOX17 cDNA which displays the positionsof conserved motifs and highlights the region used for the immunizationprocedure by GENOVAC.

FIG. 3 is a relational dendrogram illustrating that SOX17 is mostclosely related to SOX7 and somewhat less to SOX18. The SOX17 proteinsare more closely related among species homologs than to other members ofthe SOX group F subfamily within the same species.

FIG. 4 is a Western blot probed with the rat anti-SOX17 antibody. Thisblot demonstrates the specificity of this antibody for human SOX17protein over-expressed in fibroblasts (lane 1) and a lack ofimmunoreactivity with EGFP (lane 2) or the most closely related SOXfamily member, SOX7 (lane 3).

FIGS. 5A-5B are micrographs showing a cluster of SOX17⁺ cells thatdisplay a significant number of AFP⁺ co-labeled cells (A). This is instriking contrast to other SOX17⁺ clusters (B) where little or no AFP⁺cells are observed.

FIGS. 6A-6C are micrographs showing parietal endoderm and SOX17. Panel Ashows immunocytochemistry for human Thrombomodulin (TM) protein locatedon the cell surface of parietal endoderm cells in randomlydifferentiated cultures of hES cells. Panel B is the identical fieldshown in A double-labeled for TM and SOX17. Panel C is the phasecontrast image of the same field with DAPI labeled nuclei. Note thecomplete correlation of DAPI labeled nuclei and SOX17 labeling.

FIGS. 7A-7B are bar charts showing SOX17 gene expression by quantitativePCR (Q-PCR) and anti-SOX17 positive cells by SOX17-specific antibody.Panel A shows that activin A increases SOX17 gene expression whileretinoic acid (RA) strongly suppresses SOX17 expression relative to theundifferentiated control media (SR20). Panel B shows the identicalpattern as well as a similar magnitude of these changes is reflected inSOX17⁺ cell number, indicating that Q-PCR measurement of SOX17 geneexpression is very reflective of changes at the single cell level.

FIG. 8A is a bar chart which shows that a culture of differentiatinghESCs in the presence of activin A maintains a low level of AFP geneexpression while cells allowed to randomly differentiate in 10% fetalbovine serum (FBS) exhibit a strong upregulation of AFP. The differencein expression levels is approximately 7-fold.

FIGS. 8B-8C are images of two micrographs showing that the suppressionof AFP expression by activin A is also evident at the single cell levelas indicated by the very rare and small clusters of AFP⁺ cells observedin activin A treatment conditions (bottom) relative to 10% FBS alone(top).

FIGS. 9A-9B are comparative images showing the quantitation of the AFPcell number using flow cytometry. This figure demonstrates that themagnitude of change in AFP gene expression (FIG. 8A) in the presence(right panel) and absence (left panel) of activin A exactly correspondsto the number of AFP⁺ cells, further supporting the utility of Q-PCRanalyses to indicate changes occurring at the individual cell level.

FIGS. 10A-10F are micrographs which show that exposure of hESCs tonodal, activin A and activin B (NAA) yields a striking increase in thenumber of SOX17⁺ cells over the period of 5 days (A-C). By comparing tothe relative abundance of SOX17⁺ cells to the total number of cellspresent in each field, as indicated by DAPI stained nuclei (D-F), it canbe seen that approximately 30-50% of all cells are immunoreactive forSOX17 after five days treatment with NAA.

FIG. 11 is a bar chart which demonstrates that activin A (0, 10, 30 or100 ng/ml) dose-dependently increases SOX17 gene expression indifferentiating hESCs. Increased expression is already robust after 3days of treatment on adherent cultures and continues through subsequent1, 3 and 5 days of suspension culture as well.

FIGS. 12A-12C are bar charts which demonstrate the effect of activin Aon the expression of MIXL1 (panel A), GATA4 (panel B) and HNF3b (panelC). Activin A dose-dependent increases are also observed for three othermarkers of definitive endoderm; MIXL1, GATA4 and HNF3b. The magnitudesof increased expression in response to activin dose are strikinglysimilar to those observed for SOX17, strongly indicating that activin Ais specifying a population of cells that co-express all four genes(SOX17⁺, MIXL1⁺, GATA4⁺ and HNF3b⁺).

FIGS. 13A-13C are bar charts which demonstrate the effect of activin Aon the expression of AFP (panel A), SOX7 (panel B) and SPARC (panel C).There is an activin A dose-dependent decrease in expression of thevisceral endoderm marker AFP. Markers of primitive endoderm (SOX7) andparietal endoderm (SPARC) remain either unchanged or exhibit suppressionat some time points indicating that activin A does not act to specifythese extra-embryonic endoderm cell types. This further supports thefact that the increased expression of SOX17, MIXL1, GATA4, and HNF3b aredue to an increase in the number of definitive endoderm cells inresponse to activin A.

FIGS. 14A-14B are bar charts showing the effect of activin A on ZIC1(panel A) and Brachyury expression (panel B) Consistent expression ofthe neural marker ZIC1 demonstrates that there is not a dose-dependenteffect of activin A on neural differentiation. There is a notablesuppression of mesoderm differentiation mediated by 100 ng/ml of activinA treatment as indicated by the decreased expression of brachyury. Thisis likely the result of the increased specification of definitiveendoderm from the mesendoderm precursors. Lower levels of activin Atreatment (10 and 30 ng/ml) maintain the expression of brachyury atlater time points of differentiation relative to untreated controlcultures.

FIGS. 15A-15B are micrographs showing decreased parietal endodermdifferentiation in response to treatment with activins. Regions ofTM^(hi) parietal endoderm are found through the culture (A) whendifferentiated in serum alone, while differentiation to TM⁺ cells isscarce when activins are included (B) and overall intensity of TMimmunoreactivity is lower.

FIGS. 16A-16D are micrographs which show marker expression in responseto treatment with activin A and activin B. hESCs were treated for fourconsecutive days with activin A and activin B and triple labeled withSOX17, AFP and TM antibodies. Panel A—SOX17; Panel B—AFP; Panel C—TM;and Panel D—Phase/DAPI. Notice the numerous SOX17 positive cells (A)associated with the complete absence of AFP (B) and TM (C)immunoreactivity.

FIG. 17 is a micrograph showing the appearance of definitive endodermand visceral endoderm in vitro from hESCs. The regions of visceralendoderm are identified by AFP^(hi)/SOX17^(lo/-) while definitiveendoderm displays the complete opposite profile, SOX17^(hi)/AFP^(lo/-).This field was selectively chosen due to the proximity of these tworegions to each other. However, there are numerous times whenSOX17^(hi)/AFP^(lo/-) regions are observed in absolute isolation fromany regions of AFP^(hi) cells, suggesting the separate origination ofthe definitive endoderm cells from visceral endoderm cells.

FIG. 18 is a diagram depicting the TGFβ family of ligands and receptors.Factors activating AR Smads and BR Smads are useful in the production ofdefinitive endoderm from human embryonic stem cells (see, J CellPhysiol. 187:265-76).

FIG. 19 is a bar chart showing the induction of SOX17 expression overtime as a result of treatment with individual and combinations of TGFβfactors.

FIG. 20 is a bar chart showing the increase in SOX17⁺ cell number withtime as a result of treatment with combinations of TGFβ factors.

FIG. 21 is a bar chart showing induction of SOX17 expression over timeas a result of treatment with combinations of TGFβ factors.

FIG. 22 is a bar chart showing that activin A induces a dose-dependentincrease in SOX17⁺ cell number.

FIG. 23 is a bar chart showing that addition of Wnt3a to activin A andactivin B treated cultures increases SOX17 expression above the levelsinduced by activin A and activin B alone.

FIGS. 24A-24C are bar charts showing differentiation to definitiveendoderm is enhanced in low FBS conditions. Treatment of hESCs withactivins A and B in media containing 2% FBS (2AA) yields a 2-3 timesgreater level of SOX17 expression as compared to the same treatment in10% FBS media (10AA) (panel A). Induction of the definitive endodermmarker MIXL1 (panel B) is also affected in the same way and thesuppression of AFP (visceral endoderm) (panel C) is greater in 2% FBSthan in 10% FBS conditions.

FIGS. 25A-25D are micrographs which show SOX17⁺ cells are dividing inculture. SOX17 immunoreactive cells are present at the differentiatingedge of an hESC colony (C, D) and are labeled with proliferating cellnuclear antigen (PCNA) (panel B) yet are not co-labeled with OCT4 (panelC). In addition, clear mitotic figures can be seen by DAPI labeling ofnuclei in both SOX17⁺ cells (arrows) as well as OCT4⁺, undifferentiatedhESCs (arrowheads) (D).

FIG. 26 is a bar chart showing the relative expression level of CXCR4 indifferentiating hESCs under various media conditions.

FIGS. 27A-27D are bar charts that show how a panel of definitiveendoderm markers share a very similar pattern of expression to CXCR4across the same differentiation treatments displayed in FIG. 26.

FIGS. 28A-28E are bar charts showing how markers for mesoderm(BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm (SOX7)exhibit an inverse relationship to CXCR4 expression across the sametreatments displayed in FIG. 26.

FIGS. 29A-29F are micrographs that show the relative difference in SOX17immunoreactive cells across three of the media conditions displayed inFIGS. 26-28.

FIGS. 30A-30C are flow cytometry dot plots that demonstrate the increasein CXCR4⁺ cell number with increasing concentration of activin A addedto the differentiation media.

FIGS. 31A-31D are bar charts that show the CXCR4⁺ cells isolated fromthe high dose activin A treatment (A100-CX+) are even further enrichedfor definitive endoderm markers than the parent population (A100).

FIG. 32 is a bar chart showing gene expression from CXCR4⁺ and CXCR4⁻cells isolated using fluorescence-activated cell sorting (FACS) as wellas gene expression in the parent populations. This demonstrates that theCXCR4⁺ cells contain essentially all the CXCR4 gene expression presentin each parent population and the CXCR4⁻ populations contain very littleor no CXCR4 gene expression.

FIGS. 33A-33D are bar charts that demonstrate the depletion of mesoderm(BRACHYURY, MOX1), ectoderm (ZIC1) and visceral endoderm (SOX7) geneexpression in the CXCR4+ cells isolated from the high dose activin Atreatment which is already suppressed in expression of thesenon-definitive endoderm markers.

FIGS. 34A-34M are bar charts showing the expression patterns of markergenes that can be used to identify definitive endoderm cells. Theexpression analysis of definitive endoderm markers, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1 is shown in panels G-L, respectively. Theexpression analysis of previously described lineage marking genes,SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOX1 is shown in panels A-F,respectively. Panel M shows the expression analysis of CXCR4. Withrespect to each of panels A-M, the column labeled hESC indicates geneexpression from purified human embryonic stem cells; 2NF indicates cellstreated with 2% FBS, no activin addition; 0.1A100 indicates cellstreated with 0.1% FBS, 100 ng/ml activin A; 1A100 indicates cellstreated with 1% FBS, 100 ng/ml activin A; and 2A100 indicates cellstreated with 2% FBS, 100 ng/ml activin A.

FIG. 35 is a chart which shows the relative expression of the PDX1 genein a culture of hESCs after 4 days and 6 days with and without activinin the presence of retinoic acid (RA) and fibroblast growth factor(FGF-10) added on day 4.

FIGS. 36A-36F are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 6 days with and withoutactivin in the presence of retinoic acid (RA) and fibroblast growthfactor (FGF-10) added on day 4. The panels show the relative levels ofexpression of the following marker genes: (A) SOX17; (B) SOX7; (C) AFP;(D) SOX1; (E) ZIC1; and (F) NFM.

FIGS. 37A-37C are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence or absence of combinations of retinoic acid(RA), fibroblast growth factor (FGF-10) and fibroblast growth factor(FGF-4) added on day 4. The panels show the relative levels ofexpression of the following marker genes: (A) PDX1; (B) SOX7; and (C)NFM.

FIGS. 38A-38G are charts which show the relative expression of markergenes in a culture of definitive endoderm cells contacted with 50 ng/mlFGF-10 in combination with either 1 μM, 0.2 μM or 0.04 μM retinoic acid(RA) added on day 4. The panels show the relative levels of expressionof the following marker genes: (A) PDX1; (B) HOXA3; (C) HOXC6; (D)HOXA13; (E) CDX1; (F) SOX1; and (G) NFM.

FIGS. 39A-39E are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence of combinations of retinoic acid (RA),fibroblast growth factor (FGF-10) and one of the following: serumreplacement (SR), fetal bovine serum (FBS) or B27. The panels show therelative levels of expression of the following marker genes: (A) PDX1;(B) SOX7; (C) AFP; (D) ZIC1; and (E) NFM.

FIGS. 40A-40B are charts which show the relative expression of markergenes for pancreas (PDX1, HNF6) and liver (HNF6) in a culture of hESCsafter 6 days (just prior to addition of RA) and at 9 days (three daysafter exposure to RA). Various conditions were included to compare theaddition of activin B at doses of 10 ng/ml (a10), 25 ng/ml (a25) or 50ng/ml (a50) in the presence of either 25 ng/ml (A25) or 50 ng/ml (A50)activin A. The condition without any activin A or activin B (NF) servesas the negative control for definitive endoderm and PDX1-positiveendoderm production. The panels show the relative levels of expressionof the following marker genes: (A) PDX1 and (B) HNF6.

FIGS. 41A-41C are charts which show the relative expression of markergenes in a culture of hESCs with 100 ng/ml (A100), 50 ng/ml (A50) orwithout (NF) activin A at 5 days (just prior to retinoic acid addition)and at 2, 4, and 6 days after RA exposure (day 7, 9, and 11,respectively). The percentage label directly under each bar indicatesthe FBS dose during days 3-5 of differentiation. Starting at day 7,cells treated with RA (R) were grown in RPMI medium comprising 0.5% FBS.The RA concentration was 2 μM on day 7, 1 μM on day 9 and 0.2 μM on day11. The panels show the relative levels of expression of the followingmarker genes: (A) PDX1; (B) ZIC1; (C) SOX7.

FIGS. 42A-42B are charts which show the relative expression of markergenes in a culture of hESCs treated first with activin A in low FBS toinduce definitive endoderm (day 5) and then with fresh (A25R) mediumcomprising 25 ng/ml activin A and RA or various conditioned media(MEFCM, CM#2, CM#3 and CM#4) and RA to induce PDX1-expressing endoderm.Marker expression was determined on days 5, 6, 7, 8 and 9. The panelsshow the relative levels of expression of the following marker genes:(A) PDX1; (B) CDX1.

FIG. 43 is a chart which shows the relative expression of PDX1 in aculture of hESCs treated first with activin A in low FBS to inducedefinitive endoderm and followed by fresh media comprising activin A andretinoic acid (A25R) or varying amounts of RA in conditioned mediadiluted into fresh media. Total volume of media is 5 ml in all cases.

FIG. 44 is a Western blot showing PDX1 immunoprecipitated fromRA-treated definitive endoderm cells 3 days (d8) and 4 days (d9) afterthe addition of RA and 50 ng/ml activin A.

FIG. 45 is a summary chart displaying the results of afluorescence-activated cell sort (FACs) of PDX1-positive foregutendoderm cells genetically tagged with a EGFP reporter under control ofthe PDX1 promoter.

FIG. 46 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg) and EGFP-positive cells (GFP+).

FIG. 47 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg), the half of the EGFP-positive cell populationthat has the lowest EGFP signal intensity (Lo) and the half of theEGFP-positive cell population that has the highest EGFP signal intensity(Hi).

FIGS. 48A-48E are a charts showing the relative expression levelsnormalized to housekeeping genes of five pancreatic endoderm markers insorted populations of live cells (Live), EGFP-negative cells (Neg) andEGFP-positive cells (GFP+). Panels: A—NKX2.2; B—GLUT2; C—HNF313; D—KRT19and E—HNF4α.

FIGS. 49A-49B are a charts showing the relative expression levelsnormalized to housekeeping genes of two non-pancreatic endoderm markersin sorted populations of live cells (Live), EGFP-negative cells (Neg)and EGFP-positive cells (GFP+). Panels: A—ZIC1 and B—GFAP.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand when appropriate. The Sequence Listing is submitted asan ASCII text file [9511-96321-07_Sequence_Listing.txt, Oct. 30, 2018,5.78 KB], which is incorporated by reference herein.

DETAILED DESCRIPTION

A crucial stage in early human development termed gastrulation occurs2-3 weeks after fertilization. Gastrulation is extremely significantbecause it is at this time that the three primary germ layers are firstspecified and organized (Lu et al., 2001; Schoenwolf and Smith, 2000).The ectoderm is responsible for the eventual formation of the outercoverings of the body and the entire nervous system whereas the heart,blood, bone, skeletal muscle and other connective tissues are derivedfrom the mesoderm. Definitive endoderm is defined as the germ layer thatis responsible for formation of the entire gut tube which includes theesophagus, stomach and small and large intestines, and the organs whichderive from the gut tube such as the lungs, liver, thymus, parathyroidand thyroid glands, gall bladder and pancreas (Grapin-Botton and Melton,2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells andMelton, 1999; Wells and Melton, 2000). A very important distinctionshould be made between the definitive endoderm and the completelyseparate lineage of cells termed primitive endoderm. The primitiveendoderm is primarily responsible for formation of extra-embryonictissues, mainly the parietal and visceral endoderm portions of theplacental yolk sac and the extracellular matrix material of Reichert'smembrane. During gastrulation, the process of definitive endodermformation begins with a cellular migration event in which mesendodermcells (cells competent to form mesoderm or endoderm) migrate through astructure called the primitive streak. Definitive endoderm is derivedfrom cells, which migrate through the anterior portion of the streak andthrough the node (a specialized structure at the anterior-most region ofthe streak). As migration occurs, definitive endoderm populates firstthe most anterior gut tube and culminates with the formation of theposterior end of the gut tube.

The PDX1 Gene Expression During Development

PDX1 (also called STF-1, IDX-1 and IPF-1) is a transcription factor thatis necessary for development of the pancreas and rostral duodenum. PDX1is first expressed in the pancreatic endoderm, which arises fromposterior foregut endoderm and will produce both the exocrine andendocrine cells, starting at E8.5 in the mouse. Later, PDX1 becomesrestricted to beta-cells and some delta-cells. This expression patternis maintained in the adult. PDX1 is also expressed in duodenal endodermearly in development, which is adjacent to the forming pancreas, then inthe duodenal enterocytes and enteroendocrine cells, antral stomach andin the common bile, cystic and biliary ducts. This region of expressionalso becomes limited, at the time that pancreatic expression becomesrestricted, to predominantly the rostral duodenum.

PDX1-Positive Cells and Processes Related Thereto

Embodiments of the present invention relate to novel, defined processesfor the production of PDX1-positive endoderm cells, wherein thePDX1-positive endoderm cells are multipotent cells that candifferentiate into cells, tissues or organs derived from theforegut/midgut region of the gut tube (PDX1-positive foregut/midgutendoderm). As used herein, “multipotent” or “multipotent cell” refers toa cell type that can give rise to a limited number of other particularcell types. As used herein, “foregut/midgut” refers to cells of theanterior portion of the gut tube as well as cells of the middle portionof the gut tube, including cells of the foregut/midgut junction.

Some preferred embodiments of the present invention relate to processesfor the production of PDX1-positive foregut endoderm cells. In someembodiments, these PDX1-positive foregut endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe anterior portion of the gut tube (PDX1-positive foregut endoderm).

Additional preferred embodiments relate to processes for the productionof PDX1-positive endoderm cells of the posterior portion of the foregut.In some embodiments, these PDX1-positive endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe posterior portion of the foregut region of the gut tube.

The PDX1-positive foregut endoderm cells, such as those producedaccording to the methods described herein, can be used to produce fullydifferentiated insulin-producing β-cells. In some embodiments of thepresent invention, PDX1-positive foregut endoderm cells are produced bydifferentiating definitive endoderm cells that do not substantiallyexpress PDX1 (PDX1-negative definitive endoderm cells; also referred toherein as definitive endoderm) so as to form PDX1-positive foregutendoderm cells. PDX1-negative definitive endoderm cells can be preparedby differentiating pluripotent cells, such as embryonic stem cells, asdescribed herein or by any other known methods. A convenient and highlyefficient method for producing PDX1-negative definitive endoderm frompluripotent cells is described in U.S. patent Ser. No. 11/021,618,entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, the disclosure ofwhich is incorporated herein by reference in its entirety.

Processes of producing PDX1-positive foregut endoderm cells provide abasis for efficient production of pancreatic tissues such as acinarcells, ductal cells and islet cells from pluripotent cells. In certainpreferred embodiments, human PDX1-positive foregut endoderm cells arederived from human PDX1-negative definitive endoderm cells, which inturn, are derived from hESCs. These human PDX1-positive foregut endodermcells can then be used to produce functional insulin-producing β-cells.To obtain useful quantities of insulin-producing f3-cells, highefficiency of differentiation is desirable for each of thedifferentiation steps that occur prior to reaching the pancreaticislet/β-cell fate. Because differentiation of PDX1-negative definitiveendoderm cells to PDX1-positive foregut endoderm cells represents anearly step towards the production of functional pancreatic islet/β-cells(as shown in FIG. 1), high efficiency of differentiation at this step isparticularly desirable.

In view of the desirability of efficient differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells, some aspects of the present invention relate to in vitromethodology that results in approximately 2-25% conversion ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells. Typically, such methods encompass the application ofculture and growth factor conditions in a defined and temporallyspecified fashion. Further enrichment of the cell population forPDX1-positive foregut endoderm cells can be achieved by isolation and/orpurification of the PDX1-positive foregut endoderm cells from othercells in the population by using a reagent that specifically binds tothe PDX1-positive foregut endoderm cells. As an alternative,PDX1-positive foregut endoderm cells can be labeled with a reportergene, such as green fluorescent protein (GFP), so as to enable thedetection of PDX1 expression. Such fluorescently labeled cells can thenbe purified by fluorescent activated cell sorting (FACS). Furtheraspects of the present invention relate to cell cultures and enrichedcell populations comprising PDX1-positive foregut endoderm cells as wellas methods for identifying factors useful in the differentiation to andfrom PDX1-positive foregut endoderm.

In order to determine the amount of PDX1-positive foregut endoderm cellsin a cell culture or cell population, a method of distinguishing thiscell type from the other cells in the culture or in the population isdesirable. Accordingly, certain embodiments of the present inventionrelate to cell markers whose presence, absence and/or relativeexpression levels are indicative of PDX1-positive foregut endoderm cellsas well as methods for detecting and determining the expression of suchmarkers. As used herein, “expression” refers to the production of amaterial or substance as well as the level or amount of production of amaterial or substance. Thus, determining the expression of a specificmarker refers to detecting either the relative or absolute amount of themarker that is expressed or simply detecting the presence or absence ofthe marker. As used herein, “marker” refers to any molecule that can beobserved or detected. For example, a marker can include, but is notlimited to, a nucleic acid, such as a transcript of a specific gene, apolypeptide product of a gene, a non-gene product polypeptide, aglycoprotein, a carbohydrate, a glycolipd, a lipid, a lipoprotein or asmall molecule (for example, molecules having a molecular weight of lessthan 10,000 amu).

In some embodiments of the present invention, the presence, absenceand/or level of expression of a marker is determined by quantitative PCR(Q-PCR). For example, the amount of transcript produced by certaingenetic markers, such as PDX1, SOX17, SOX7, SOX1, ZIC1, NFM,alpha-fetoprotein (AFP), homeobox A13 (HOXA13), homeobox C6 (HOXC6),and/or other markers described herein is determined by Q-PCR. In otherembodiments, immunohistochemistry is used to detect the proteinsexpressed by the above-mentioned genes. In still other embodiments,Q-PCR and immunohistochemical techniques are both used to identify anddetermine the amount or relative proportions of such markers.

By using the differentiation and detection methods described herein, itis possible to identify PDX1-positive foregut endoderm cells, as well asdetermine the proportion of PDX1-positive foregut endoderm cells in acell culture or cell population. For example, in some embodiments of thepresent invention, the PDX1-positive foregut endoderm cells or cellpopulations that are produced express the PDX1 gene at a level of atleast about 2 orders of magnitude greater than PDX1-negative cells orcell populations. In other embodiments, the PDX1-positive foregutendoderm cells and cell populations that are produced express the PDX1gene at a level of more than 2 orders of magnitude greater thanPDX1-negative cells or cell populations. In still other embodiments, thePDX1-positive foregut endoderm cells or cell populations that areproduced express one or more of the markers selected from the groupconsisting of PDX1, SOX17, HOXA13 and HOXC6 at a level of about 2 ormore than 2 orders of magnitude greater than PDX1-negative definitiveendoderm cells or cell populations.

The compositions and methods described herein have several usefulfeatures. For example, the cell cultures and cell populations comprisingPDX1-positive endoderm, as well as the methods for producing such cellcultures and cell populations, are useful for modeling the early stagesof human development. Furthermore, the compositions and methodsdescribed herein can also serve for therapeutic intervention in diseasestates, such as diabetes mellitus. For example, since PDX1-positiveforegut endoderm serves as the source for only a limited number oftissues, it can be used in the development of pure tissue or cell types.

Production of PDX1-Negative Definitive Endoderm (Definitive Endoderm)from Pluripotent Cells

Cell cultures and/or cell populations comprising PDX1-positive foregutendoderm cells are produced from pluripotent cells by first producingPDX1-negative definitive endoderm (also referred to as “definitiveendoderm”). Processes for differentiating pluripotent cells to producecell cultures and enriched cell populations comprising definitiveendoderm is described briefly below and in detail in U.S. patent Ser.No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosure of which is incorporated herein by reference in its entirety.In some of these processes, the pluripotent cells used as startingmaterial are stem cells. In certain processes, definitive endoderm cellcultures and enriched cell populations comprising definitive endodermcells are produced from embryonic stem cells. As used herein,“embryonic” refers to a range of developmental stages of an organismbeginning with a single zygote and ending with a multicellular structurethat no longer comprises pluripotent or totipotent cells other thandeveloped gametic cells. In addition to embryos derived by gametefusion, the term “embryonic” refers to embryos derived by somatic cellnuclear transfer. A preferred method for deriving definitive endodermcells utilizes human embryonic stem cells as the starting material fordefinitive endoderm production. Such pluripotent cells can be cells thatoriginate from the morula, embryonic inner cell mass or those obtainedfrom embryonic gonadal ridges. Human embryonic stem cells can bemaintained in culture in a pluripotent state without substantialdifferentiation using methods that are known in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,453,357, 5,670,372,5,690,926 5,843,780, 6,200,806 and 6,251,671 the disclosures of whichare incorporated herein by reference in their entireties.

In some processes for producing definitive endoderm cells, hESCs aremaintained on a feeder layer. In such processes, any feeder layer whichallows hESCs to be maintained in a pluripotent state can be used. Onecommonly used feeder layer for the cultivation of human embryonic stemcells is a layer of mouse fibroblasts. More recently, human fibroblastfeeder layers have been developed for use in the cultivation of hESCs(see US Patent Application No. 2002/0072117, the disclosure of which isincorporated herein by reference in its entirety). Alternative processesfor producing definitive endoderm permit the maintenance of pluripotenthESC without the use of a feeder layer. Methods of maintainingpluripotent hESCs under feeder-free conditions have been described in USPatent Application No. 2003/0175956, the disclosure of which isincorporated herein by reference in its entirety.

The human embryonic stem cells used herein can be maintained in cultureeither with or without serum. In some embryonic stem cell maintenanceprocedures, serum replacement is used. In others, serum free culturetechniques, such as those described in US Patent Application No.2003/0190748, the disclosure of which is incorporated herein byreference in its entirety, are used.

Stem cells are maintained in culture in a pluripotent state by routinepassage until it is desired that they be differentiated into definitiveendoderm. In some processes, differentiation to definitive endoderm isachieved by providing to the stem cell culture a growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation todefinitive endoderm. Growth factors of the TGFβ superfamily which areuseful for the production of definitive endoderm are selected from theNodal/Activin or BMP subgroups. In some preferred differentiationprocesses, the growth factor is selected from the group consisting ofNodal, activin A, activin B and BMP4. Additionally, the growth factorWnt3a and other Wnt family members are useful for the production ofdefinitive endoderm cells. In certain differentiation processes,combinations of any of the above-mentioned growth factors can be used.

With respect to some of the processes for the differentiation ofpluripotent stem cells to definitive endoderm cells, the above-mentionedgrowth factors are provided to the cells so that the growth factors arepresent in the cultures at concentrations sufficient to promotedifferentiation of at least a portion of the stem cells to definitiveendoderm cells. In some processes, the above-mentioned growth factorsare present in the cell culture at a concentration of at least about 5ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at leastabout 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, atleast about 500 ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml, at leastabout 5000 ng/ml or more than about 5000 ng/ml.

In certain processes for the differentiation of pluripotent stem cellsto definitive endoderm cells, the above-mentioned growth factors areremoved from the cell culture subsequent to their addition. For example,the growth factors can be removed within about one day, about two days,about three days, about four days, about five days, about six days,about seven days, about eight days, about nine days or about ten daysafter their addition. In a preferred processes, the growth factors areremoved about four days after their addition.

Cultures of definitive endoderm cells can be grown in medium containingreduced serum or no serum. Under certain culture conditions, serumconcentrations can range from about 0.05% v/v to about 20% v/v. Forexample, in some differentiation processes, the serum concentration ofthe medium can be less than about 0.05% (v/v), less than about 0.1%(v/v), less than about 0.2% (v/v), less than about 0.3% (v/v), less thanabout 0.4% (v/v), less than about 0.5% (v/v), less than about 0.6%(v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less thanabout 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v),less than about 3% (v/v), less than about 4% (v/v), less than about 5%(v/v), less than about 6% (v/v), less than about 7% (v/v), less thanabout 8% (v/v), less than about 9% (v/v), less than about 10% (v/v),less than about 15% (v/v) or less than about 20% (v/v). In someprocesses, definitive endoderm cells are grown without serum or withserum replacement. In still other processes, definitive endoderm cellsare grown in the presence of B27. In such processes, the concentrationof B27 supplement can range from about 0.1% v/v to about 20% v/v.

Monitoring the Differentiation of Pluripotent Cells to PDX1-NegativeDefinitive Endoderm (Definitive Endoderm)

The progression of the hESC culture to definitive endoderm can bemonitored by determining the expression of markers characteristic ofdefinitive endoderm. In some processes, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such processes, themeasurement of marker expression can be qualitative or quantitative. Onemethod of quantitating the expression of markers that are produced bymarker genes is through the use of quantitative PCR (Q-PCR). Methods ofperforming Q-PCR are well known in the art. Other methods which areknown in the art can also be used to quantitate marker gene expression.For example, the expression of a marker gene product can be detected byusing antibodies specific for the marker gene product of interest. Incertain processes, the expression of marker genes characteristic ofdefinitive endoderm as well as the lack of significant expression ofmarker genes characteristic of hESCs and other cell types is determined.

As described further in the Examples below, a reliable marker ofdefinitive endoderm is the SOX17 gene. As such, the definitive endodermcells produced by the processes described herein express the SOX17marker gene, thereby producing the SOX17 gene product. Other markers ofdefinitive endoderm are MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theSOX17 marker gene at a level higher than that of the SOX7 marker gene,which is characteristic of primitive and visceral endoderm (see Table1), in some processes, the expression of both SOX17 and SOX7 ismonitored. In other processes, expression of the both the SOX17 markergene and the OCT4 marker gene, which is characteristic of hESCs, ismonitored. Additionally, because definitive endoderm cells express theSOX17 marker gene at a level higher than that of the AFP, SPARC orThrombomodulin (TM) marker genes, the expression of these genes can alsobe monitored.

Another marker of definitive endoderm is the CXCR4 gene. The CXCR4 geneencodes a cell surface chemokine receptor whose ligand is thechemoattractant SDF-1. The principal roles of the CXCR4 receptor-bearingcells in the adult are believed to be the migration of hematopoeticcells to the bone marrow, lymphocyte trafficking and the differentiationof various B cell and macrophage blood cell lineages [Kim, C., andBroxmeyer, H. J. Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptoralso functions as a coreceptor for the entry of HIV-1 into T-cells[Feng, Y., et al. Science, 272, 872-877 (1996)]. In an extensive seriesof studies carried out by [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)], the expression of the chemokine receptor CXCR4 and itsunique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte Biol. 65,6-15 (1999)], were delineated during early development and adult life inthe mouse. The CXCR4/SDF1 interaction in development became apparentwhen it was demonstrated that if either gene was disrupted in transgenicmice [Nagasawa et al. Nature, 382, 635-638 (1996)], Ma, Q., et alImmunity, 10, 463-471 (1999)] it resulted in late embryonic lethality.McGrath et al. demonstrated that CXCR4 is the most abundant chemokinereceptor messenger RNA detected during early gastrulating embryos (E7.5)using a combination of RNase protection and in situ hybridizationmethodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling appearsto be mainly involved in inducing migration of primitive-streakgermlayer cells and is expressed on definitive endoderm, mesoderm andextraembryonic mesoderm present at this time. In E7.2-7.8 mouse embryos,CXCR4 and alpha-fetoprotein are mutually exclusive indicating a lack ofexpression in visceral endoderm [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)].

Since definitive endoderm cells produced by differentiating pluripotentcells express the CXCR4 marker gene, expression of CXCR4 can bemonitored in order to track the production of definitive endoderm cells.Additionally, definitive endoderm cells produced by the methodsdescribed herein express other markers of definitive endoderm including,but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theCXCR4 marker gene at a level higher than that of the SOX7 marker gene,the expression of both CXCR4 and SOX7 can be monitored. In otherprocesses, expression of the both the CXCR4 marker gene and the OCT4marker gene, is monitored. Additionally, because definitive endodermcells express the CXCR4 marker gene at a level higher than that of theAFP, SPARC or Thrombomodulin (TM) marker genes, the expression of thesegenes can also be monitored.

It will be appreciated that expression of CXCR4 in endodermal cells doesnot preclude the expression of SOX17. As such, definitive endoderm cellsproduced by the processes described herein will substantially expressSOX17 and CXCR4 but will not substantially express AFP, TM, SPARC orPDX1.

Enrichment, Isolation and/or Purification of Definitive Endoderm

Definitive endoderm cells produced by any of the above-describedprocesses can be enriched, isolated and/or purified by using an affinitytag that is specific for such cells. Examples of affinity tags specificfor definitive endoderm cells are antibodies, ligands or other bindingagents that are specific to a marker molecule, such as a polypeptide,that is present on the cell surface of definitive endoderm cells butwhich is not substantially present on other cell types that would befound in a cell culture produced by the methods described herein. Insome processes, an antibody which binds to CXCR4 is used as an affinitytag for the enrichment, isolation or purification of definitive endodermcells. In other processes, the chemokine SDF-1 or other molecules basedon SDF-1 can also be used as affinity tags. Such molecules include, butnot limited to, SDF-1 fragments, SDF-1 fusions or SDF-1 mimetics.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and definitive endoderm cells described herein. In oneprocess, an antibody which binds to CXCR4 is attached to a magnetic beadand then allowed to bind to definitive endoderm cells in a cell culturewhich has been enzymatically treated to reduce intercellular andsubstrate adhesion. The cell/antibody/bead complexes are then exposed toa movable magnetic field which is used to separate bead-bound definitiveendoderm cells from unbound cells. Once the definitive endoderm cellsare physically separated from other cells in culture, the antibodybinding is disrupted and the cells are replated in appropriate tissueculture medium.

Additional methods for obtaining enriched, isolated or purifieddefinitive endoderm cell cultures or populations can also be used. Forexample, in some embodiments, the CXCR4 antibody is incubated with adefinitive endoderm-containing cell culture that has been treated toreduce intercellular and substrate adhesion. The cells are then washed,centrifuged and resuspended. The cell suspension is then incubated witha secondary antibody, such as an FITC-conjugated antibody that iscapable of binding to the primary antibody. The cells are then washed,centrifuged and resuspended in buffer. The cell suspension is thenanalyzed and sorted using a fluorescence activated cell sorter (FACS).CXCR4-positive cells are collected separately from CXCR4-negative cells,thereby resulting in the isolation of such cell types. If desired, theisolated cell compositions can be further purified by using an alternateaffinity-based method or by additional rounds of sorting using the sameor different markers that are specific for definitive endoderm.

In still other processes, definitive endoderm cells are enriched,isolated and/or purified using a ligand or other molecule that binds toCXCR4. In some processes, the molecule is SDF-1 or a fragment, fusion ormimetic thereof.

In preferred processes, definitive endoderm cells are enriched, isolatedand/or purified from other non-definitive endoderm cells after the stemcell cultures are induced to differentiate towards the definitiveendoderm lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the procedures just described, definitive endoderm cellsmay also be isolated by other techniques for cell isolation.Additionally, definitive endoderm cells may also be enriched or isolatedby methods of serial subculture in growth conditions which promote theselective survival or selective expansion of the definitive endodermcells.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of definitive endoderm cells and or tissues can be producedin vitro from pluripotent cell cultures or cell populations, such asstem cell cultures or populations, which have undergone at least somedifferentiation. In some methods, the cells undergo randomdifferentiation. In a preferred method, however, the cells are directedto differentiate primarily into definitive endoderm. Some preferredenrichment, isolation and/or purification methods relate to the in vitroproduction of definitive endoderm from human embryonic stem cells. Usingthe methods described herein, cell populations or cell cultures can beenriched in definitive endoderm content by at least about 2- to about1000-fold as compared to untreated cell populations or cell cultures.

Compositions Comprising PDX1-Negative Definitive Endoderm (DefinitiveEndoderm)

Cell compositions produced by the above-described methods include cellcultures comprising definitive endoderm and cell populations enriched indefinitive endoderm. For example, cell cultures which comprisedefinitive endoderm cells, wherein at least about 50-80% of the cells inculture are definitive endoderm cells, can be produced. Because theefficiency of the differentiation process can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In processes in which isolation of definitive endoderm cellsis employed, for example, by using an affinity reagent that binds to theCXCR4 receptor, a substantially pure definitive endoderm cell populationcan be recovered.

Production of PDX1-Positive Foregut Endoderm from PDX1-NegativeDefinitive Endoderm

The PDX1-positive foregut endoderm cell cultures and populationscomprising PDX1-positive foregut endoderm cells that are describedherein are produced from PDX1-negative definitive endoderm, which isgenerated from pluripotent cells as described above. A preferred methodutilizes human embryonic stem cells as the starting material. In oneembodiment, hESCs are first converted to PDX1-negative definitiveendoderm cells, which are then converted to PDX1-positive foregutendoderm cells. It will be appreciated, however, that the startingmaterials for the production of PDX1-positive foregut endoderm is notlimited to definitive endoderm cells produced using pluripotent celldifferentiation methods. Rather, any PDX1-negative definitive endodermcells can be used in the methods described herein regardless of theirorigin.

In some embodiments of the present invention, cell cultures or cellpopulations comprising PDX1-negative definitive endoderm cells can beused for further differentiation to cell cultures and/or enriched cellpopulations comprising PDX1-positive foregut endoderm cells. Forexample, a cell culture or cell population comprising humanPDX1-negative, SOX17-positive definitive endoderm cells can be used. Insome embodiments, the cell culture or cell population may also comprisedifferentiation factors, such as activins, nodals and/or BMPs, remainingfrom the previous differentiation step (that is, the step ofdifferentiating pluripotent cells to definitive endoderm cells). Inother embodiments, factors utilized in the previous differentiation stepare removed from the cell culture or cell population prior to theaddition of factors used for the differentiation of the PDX1-negative,SOX17-positive definitive endoderm cells to PDX1-positive foregutendoderm cells. In other embodiments, cell populations enriched forPDX1-negative, SOX17-positive definitive endoderm cells are used as asource for the production of PDX1-positive foregut endoderm cells.

PDX1-negative definitive endoderm cells in culture are differentiated toPDX1-positive endoderm cells by providing to a cell culture comprisingPDX1-negative, SOX17-positive definitive endoderm cells adifferentiation factor that promotes differentiation of the cells toPDX1-positive foregut endoderm cells (foregut differentiation factor).In some embodiments of the present invention, the foregutdifferentiation factor is retinoid, such as retinoic acid (RA). In someembodiments, the retinoid is used in conjunction with a fibroblastgrowth factor, such as FGF-4 or FGF-10. In other embodiments, theretinoid is used in conjunction with a member of the TGFβ superfamily ofgrowth factors and/or a conditioned medium.

By “conditioned medium” is meant, a medium that is altered as comparedto a base medium. For example, the conditioning of a medium may causemolecules, such as nutrients and/or growth factors, to be added to ordepleted from the original levels found in the base medium. In someembodiments, a medium is conditioned by allowing cells of certain typesto be grown or maintained in the medium under certain conditions for acertain period of time. For example, a medium can be conditioned byallowing hESCs to be expanded, differentiated or maintained in a mediumof defined composition at a defined temperature for a defined number ofhours. As will be appreciated by those of skill in the art, numerouscombinations of cells, media types, durations and environmentalconditions can be used to produce nearly an infinite array ofconditioned media. In some embodiments of the present invention, amedium is conditioned by allowing differentiated pluripotent cells to begrown or maintained in a medium comprising about 1% to about 20% serumconcentration. In other embodiments, a medium is conditioned by allowingdifferentiated pluripotent cells to be grown or maintained in a mediumcomprising about 1 ng/ml to about 1000 ng/ml activin A. In still otherembodiments, a medium is conditioned allowing differentiated pluripotentcells to be grown or maintained in a medium comprising about 1 ng/ml toabout 1000 ng/ml BMP4. In a preferred embodiment, a conditioned mediumis prepared by allowing differentiated hESCs to be grown or maintainedfor 24 hours in a medium, such as RPMI, comprising about 25 ng/mlactivin A and about 2 μM RA.

In some embodiments of the present invention, the cells used tocondition the medium, which is used to enhance the differentiation ofPDX1-negative definitive endoderm to PDX1-positive foregut endoderm, arecells that are differentiated from pluripotent cells, such as hESCs,over about a 5 day time period in a medium such as RPMI comprising about0% to about 20% serum and/or one or more growth/differentiation factorsof the TGFβ superfamily. Differentiation factors, such as activin A andBMP4 are supplied at concentrations ranging from about 1 ng/ml to about1000 ng/ml. In certain embodiments of the present invention, the cellsused to condition the medium are differentiated from hESCs over about a5 day period in low serum RPMI. According to some embodiments, low serumRPMI refers to a low serum containing medium, wherein the serumconcentration is gradually increased over a defined time period. Forexample, in one embodiment, low serum RPMI comprises a concentration ofabout 0.2% fetal bovine serum (FBS) on the first day of cell growth,about 0.5% FBS on the second day of cell growth and about 2% FBS on thethird through fifth day of cell growth. In another embodiment, low serumRPMI comprises a concentration of about 0% on day one, about 0.2% on daytwo and about 2% on days 3-6. In certain preferred embodiments, lowserum RPMI is supplemented with one or more differentiation factors,such as activin A and BMP4. In addition to its use in preparing cellsused to condition media, low serum RPMI can be used as a medium for thedifferentiation of PDX1-positive foregut endoderm cells fromPDX1-negative definitive endoderm cells.

It will be appreciated by those of ordinary skill in the art thatconditioned media can be prepared from media other than RPMI providedthat such media do not interfere with the growth or maintenance ofPDX1-positive foregut endoderm cells. It will also be appreciated thatthe cells used to condition the medium can be of various types. Inembodiments where freshly differentiated cells are used to condition amedium, such cells can be differentiated in a medium other than RPMIprovided that the medium does not inhibit the growth or maintenance ofsuch cells. Furthermore, a skilled artisan will appreciate that neitherthe duration of conditioning nor the duration of preparation of cellsused for conditioning is required to be 24 hours or 5 days,respectively, as other time periods will be sufficient to achieve theeffects reported herein.

In general, the use of a retinoid in combination with a fibroblastgrowth factor, a member of the TGFβ superfamily of growth factors, aconditioned medium or a combination of any of these foregutdifferentiation factors causes greater differentiation of PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm than the use of aretinoid alone. In a preferred embodiment, RA and FGF-10 are bothprovided to the PDX1-negative definitive endoderm cell culture. Inanother preferred embodiment, PDX1-negative definitive endoderm cellsare differentiated in a culture comprising a conditioned medium, activinA, activin B and RA.

With respect to some of the embodiments of differentiation processesdescribed herein, the above-mentioned foregut differentiation factorsare provided to the cells so that these factors are present in the cellculture or cell population at concentrations sufficient to promotedifferentiation of at least a portion of the PDX1-negative definitiveendoderm cell culture or cell population to PDX1-positive foregutendoderm cells. When used in connection with cell cultures and/or cellpopulations, the term “portion” means any non-zero amount of the cellculture or cell population, which ranges from a single cell to theentirety of the cell culture or cells population.

In some embodiments of the present invention, a retinoid is provided tothe cells of a cell culture such that it is present at a concentrationof at least about 0.01 μM, at least about 0.02 μM, at least about 0.04μM, at least about 0.08 μM, at least about 0.1 μM, at least about 0.2μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM,at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, atleast about 0.9 μM, at least about 1 μM, at least about 1.1 μM, at leastabout 1.2 μM, at least about 1.3 μM, at least about 1.4 μM, at leastabout 1.5 μM, at least about 1.6 μM, at least about 1.7 μM, at leastabout 1.8 μM, at least about 1.9 μM, at least about 2 μM, at least about2.1 μM, at least about 2.2 μM, at least about 2.3 μM, at least about 2.4μM, at least about 2.5 μM, at least about 2.6 μM, at least about 2.7 μM,at least about 2.8 μM, at least about 2.9 μM, at least about 3 μM, atleast about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at leastabout 5 μM, at least about 10 μM, at least about 20 μM, at least about30 μM, at least about 40 μM or at least about 50 μM. As used herein,“retinoid” refers to retinol, retinal or retinoic acid as well asderivatives of any of these compounds. In a preferred embodiment, theretinoid is retinoic acid.

In other embodiments of the present invention, one or moredifferentiation factors of the fibroblast growth factor family arepresent in the cell culture. For example, in some embodiments, FGF-4 canbe present in the cell culture at a concentration of at least about 10ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, at least about75 ng/ml, at least about 100 ng/ml, at least about 200 ng/ml, at leastabout 300 ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, orat least about 1000 ng/ml. In further embodiments of the presentinvention, FGF-10 is present in the cell culture at a concentration ofat least about 10 ng/ml, at least about 25 ng/ml, at least about 50ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least about200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at leastabout 500 ng/ml, or at least about 1000 ng/ml. In some embodiments,either FGF-4 or FGF-10, but not both, is provided to the cell culturealong with RA. In a preferred embodiment, RA is present in the cellculture at 1 μM and FGF-10 is present at a concentration of 50 ng/ml.

In some embodiments of the present invention, growth factors of the TGFβsuperfamily and/or a conditioned medium are present in the cell culture.These differentiation factors can be used in combination with RA and/orother mid-foregut differentiation factors including, but not limited to,FGF-4 and FGF-10. For example, in some embodiments, activin A and/oractivin B can be present in the cell culture at a concentration of atleast about 5 ng/ml, at least about 10 ng/ml, at least about 25 ng/ml,at least about 50 ng/ml, at least about 75 ng/ml, at least about 100ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at leastabout 400 ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.In further embodiments of the present invention, a conditioned medium ispresent in the cell culture at a concentration of at least about 1%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 100% of the total medium. In some embodiments, activin A, activinB and a conditioned medium are provided to the cell culture along withRA. In a preferred embodiment, PDX1-negative definitive endoderm cellsare differentiated to PDX1-positive foregut endoderm cells in culturescomprising about 1 μM RA, about 25 ng/ml activin A and low serum RPMImedium that has been conditioned for about 24 hours by differentiatedhESCs, wherein the differentiated hESCs have been differentiated forabout 5 days in low serum RPMI comprising about 100 ng/ml activin A. Inanother preferred embodiment, activin B and/or FGF-10 are also presentin the culture at 25 ng/ml and 50 ng/ml, respectively.

In certain embodiments of the present invention, the above-mentionedforegut differentiation factors are removed from the cell culturesubsequent to their addition. For example, the foregut differentiationfactors can be removed within about one day, about two days, about threedays, about four days, about five days, about six days, about sevendays, about eight days, about nine days or about ten days after theiraddition.

Cultures of PDX1-positive foregut endoderm cells can be grown in amedium containing reduced serum. Serum concentrations can range fromabout 0.05% (v/v) to about 20% (v/v). In some embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement. For example, incertain embodiments, the serum concentration of the medium can be lessthan about 0.05% (v/v), less than about 0.1% (v/v), less than about 0.2%(v/v), less than about 0.3% (v/v), less than about 0.4% (v/v), less thanabout 0.5% (v/v), less than about 0.6% (v/v), less than about 0.7%(v/v), less than about 0.8% (v/v), less than about 0.9% (v/v), less thanabout 1% (v/v), less than about 2% (v/v), less than about 3% (v/v), lessthan about 4% (v/v), less than about 5% (v/v), less than about 6% (v/v),less than about 7% (v/v), less than about 8% (v/v), less than about 9%(v/v), less than about 10% (v/v), less than about 15% (v/v) or less thanabout 20% (v/v). In some embodiments, PDX1-positive foregut endodermcells are grown without serum. In other embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement.

In still other embodiments, PDX1-positive foregut endoderm cells aregrown in the presence of B27. In such embodiments, B27 can be providedto the culture medium in concentrations ranging from about 0.1% (v/v) toabout 20% (v/v) or in concentrations greater than about 20% (v/v). Incertain embodiments, the concentration of B27 in the medium is about0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4%(v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v),about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20% (v/v).Alternatively, the concentration of the added B27 supplement can bemeasured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a SOX stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 50×B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toPDX1-Positive Endoderm

As with the differentiation of definitive endoderm cells frompluripotent cells, the progression of differentiation fromPDX1-negative, SOX17-positive definitive endoderm to PDX1-positiveforegut endoderm can be monitored by determining the expression ofmarkers characteristic of these cell types. Such monitoring permits oneto determine the amount of time that is sufficient for the production ofa desired amount of PDX1-positive foregut endoderm under variousconditions, for example, one or more differentiation factorconcentrations and environmental conditions. In preferred embodiments,the amount of time that is sufficient for the production of a desiredamount of PDX1-positive foregut endoderm is determined by detecting theexpression of PDX1. In some embodiments of the present invention, theexpression of certain markers is determined by detecting the presence orabsence of the marker. Alternatively, the expression of certain markerscan be determined by measuring the level at which the marker is presentin the cells of the cell culture or cell population. In suchembodiments, the measurement of marker expression can be qualitative orquantitative. As described above, a preferred method of quantitating theexpression markers that are produced by marker genes is through the useof Q-PCR. In particular embodiments, Q-PCR is used to monitor theprogression of cells of the PDX1-negative, SOX17-positive definitiveendoderm culture to PDX1-positive foregut endoderm cells by quantitatingexpression of marker genes characteristic of PDX1-positive foregutendoderm and the lack of expression of marker genes characteristic ofother cell types. Other methods which are known in the art can also beused to quantitate marker gene expression. For example, the expressionof a marker gene product can be detected by using antibodies specificfor the marker gene product of interest. In some embodiments of thepresent invention, the expression of marker genes characteristic ofPDX1-positive foregut endoderm as well as the lack of significantexpression of marker genes characteristic of PDX1-negative definitiveendoderm, hESCs and other cell types is determined.

As described further in the Examples below, PDX1 is a marker gene thatis associated with PDX1-positive foregut endoderm. As such, in someembodiments of the present invention, the expression of PDX1 isdetermined. In other embodiments, the expression of other markers, whichare expressed in PDX1-positive foregut endoderm, including, but notlimited to, SOX17, HOXA13 and/or HOXC6 is also determined. Since PDX1can also be expressed by certain other cell types (that is, visceralendoderm and certain neural ectoderm), some embodiments of the presentinvention relate to demonstrating the absence or substantial absence ofmarker gene expression that is associated with visceral endoderm and/orneural ectoderm. For example, in some embodiments, the expression ofmarkers, which are expressed in visceral endoderm and/or neural cells,including, but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM isdetermined.

In some embodiments, PDX1-positive foregut endoderm cell culturesproduced by the methods described herein are substantially free of cellsexpressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certainembodiments, the PDX1-positive foregut endoderm cell cultures producedby the processes described herein are substantially free of visceralendoderm, parietal endoderm and/or neural cells.

Enrichment, Isolation and/or Purification of PDX1-Positive ForegutEndoderm

With respect to additional aspects of the present invention,PDX1-positive foregut endoderm cells can be enriched, isolated and/orpurified. In some embodiments of the present invention, cell populationsenriched for PDX1-positive foregut endoderm cells are produced byisolating such cells from cell cultures.

In some embodiments of the present invention, PDX1-positive foregutendoderm cells are fluorescently labeled then isolated from non-labeledcells by using a fluorescence activated cell sorter (FACS). In suchembodiments, a nucleic acid encoding green fluorescent protein (GFP) oranother nucleic acid encoding an expressible fluorescent marker gene isused to label PDX1-positive cells. For example, in some embodiments, atleast one copy of a nucleic acid encoding GFP or a biologically activefragment thereof is introduced into a pluripotent cell, preferably ahuman embryonic stem cell, downstream of the PDX1 promoter such that theexpression of the GFP gene product or biologically active fragmentthereof is under control of the PDX1 promoter. In some embodiments, theentire coding region of the nucleic acid, which encodes PDX1, isreplaced by a nucleic acid encoding GFP or a biologically activefragment thereof. In other embodiments, the nucleic acid encoding GFP ora biologically active fragment thereof is fused in frame with at least aportion of the nucleic acid encoding PDX1, thereby generating a fusionprotein. In such embodiments, the fusion protein retains a fluorescentactivity similar to GFP.

Fluorescently marked cells, such as the above-described pluripotentcells, are differentiated to definitive endoderm and then toPDX1-positive foregut endoderm as described previously above. BecausePDX1-positive foregut endoderm cells express the fluorescent markergene, whereas PDX1-negative cells do not, these two cell types can beseparated. In some embodiments, cell suspensions comprising a mixture offluorescently-labeled PDX1-positive cells and unlabeled PDX1-negativecells are sorted using a FACS. PDX1-positive cells are collectedseparately from PDX1-negative cells, thereby resulting in the isolationof such cell types. If desired, the isolated cell compositions can befurther purified by additional rounds of sorting using the same ordifferent markers that are specific for PDX1-positive foregut endoderm.

In addition to the procedures just described, PDX1-positive foregutendoderm cells may also be isolated by other techniques for cellisolation. Additionally, PDX1-positive foregut endoderm cells may alsobe enriched or isolated by methods of serial subculture in growthconditions which promote the selective survival or selective expansionof said PDX1-positive foregut endoderm cells.

It will be appreciated that the above-described enrichment, isolationand purification procedures can be used with such cultures at any stageof differentiation.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of PDX1-positive foregut endoderm cells and/or tissues canbe produced in vitro from PDX1-negative, SOX17-positive definitiveendoderm cell cultures or cell populations which have undergone at leastsome differentiation. In some embodiments, the cells undergo randomdifferentiation. In a preferred embodiment, however, the cells aredirected to differentiate primarily into PDX1-positive foregut endodermcells. Some preferred enrichment, isolation and/or purification methodsrelate to the in vitro production of PDX1-positive foregut endodermcells from human embryonic stem cells.

Using the methods described herein, cell populations or cell culturescan be enriched in PDX1-positive foregut endoderm cell content by atleast about 2- to about 1000-fold as compared to untreated cellpopulations or cell cultures. In some embodiments, PDX1-positive foregutendoderm cells can be enriched by at least about 5- to about 500-fold ascompared to untreated cell populations or cell cultures. In otherembodiments, PDX1-positive foregut endoderm cells can be enriched fromat least about 10- to about 200-fold as compared to untreated cellpopulations or cell cultures. In still other embodiments, PDX1-positiveforegut endoderm cells can be enriched from at least about 20- to about100-fold as compared to untreated cell populations or cell cultures. Inyet other embodiments, PDX1-positive foregut endoderm cells can beenriched from at least about 40- to about 80-fold as compared tountreated cell populations or cell cultures. In certain embodiments,PDX1-positive foregut endoderm cells can be enriched from at least about2- to about 20-fold as compared to untreated cell populations or cellcultures.

Compositions Comprising PDX1-Positive Foregut Endoderm

Some embodiments of the present invention relate to cell compositions,such as cell cultures or cell populations, comprising PDX1-positiveendoderm cells, wherein the PDX1-positive endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe anterior portion of the gut tube (PDX1-positive foregut endoderm).In accordance with certain embodiments, the PDX1-positive foregutendoderm are mammalian cells, and in a preferred embodiment, thedefinitive endoderm cells are human cells.

Other embodiments of the present invention relate to compositions, suchas cell cultures or cell populations, comprising cells of one or morecell types selected from the group consisting of hESCs, PDX1-negativedefinitive endoderm cells, PDX1-positive foregut endoderm cells andmesoderm cells. In some embodiments, hESCs comprise less than about 5%,less than about 4%, less than about 3%, less than about 2% or less thanabout 1% of the total cells in the culture. In other embodiments,PDX1-negative definitive endoderm cells comprise less than about 90%,less than about 85%, less than about 80%, less than about 75%, less thanabout 70%, less than about 65%, less than about 60%, less than about55%, less than about 50%, less than about 45%, less than about 40%, lessthan about 35%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 12%, less than about10%, less than about 8%, less than about 6%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2% or less than about1% of the total cells in the culture. In yet other embodiments, mesodermcells comprise less than about 90%, less than about 85%, less than about80%, less than about 75%, less than about 70%, less than about 65%, lessthan about 60%, less than about 55%, less than about 50%, less thanabout 45%, less than about 40%, less than about 35%, less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 12%, less than about 10%, less than about 8%, less than about6%, less than about 5%, less than about 4%, less than about 3%, lessthan about 2% or less than about 1% of the total cells in the culture.

Additional embodiments of the present invention relate to compositions,such as cell cultures or cell populations, produced by the processesdescribed herein comprise PDX1-positive foregut endoderm as the majoritycell type. In some embodiments, the processes described herein producecell cultures and/or cell populations comprising at least about 99%, atleast about 98%, at least about 97%, at least about 96%, at least about95%, at least about 94%, at least about 93%, at least about 92%, atleast about 91%, at least about 90%, at least about 85%, at least about80%, at least about 75%, at least about 70%, at least about 65%, atleast about 60%, at least about 55%, at least about 54%, at least about53%, at least about 52% or at least about 51% PDX1-positive foregutendoderm cells. In preferred embodiments the cells of the cell culturesor cell populations comprise human cells. In other embodiments, theprocesses described herein produce cell cultures or cell populationscomprising at least about 50%, at least about 45%, at least about 40%,at least about 35%, at least about 30%, at least about 25%, at leastabout 24%, at least about 23%, at least about 22%, at least about 21%,at least about 20%, at least about 19%, at least about 18%, at leastabout 17%, at least about 16%, at least about 15%, at least about 14%,at least about 13%, at least about 12%, at least about 11%, at leastabout 10%, at least about 9%, at least about 8%, at least about 7%, atleast about 6%, at least about 5%, at least about 4%, at least about 3%,at least about 2% or at least about 1% PDX1-positive foregut endodermcells. In preferred embodiments, the cells of the cell cultures or cellpopulations comprise human cells. In some embodiments, the percentage ofPDX1-positive foregut endoderm cells in the cell cultures or populationsis calculated without regard to the feeder cells remaining in theculture.

Still other embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mixtures ofPDX1-positive foregut endoderm cells and PDX1-negative definitiveendoderm cells. For example, cell cultures or cell populationscomprising at least about 5 PDX1-positive foregut endoderm cells forabout every 95 PDX1-negative definitive endoderm cells can be produced.In other embodiments, cell cultures or cell populations comprising atleast about 95 PDX1-positive foregut endoderm cells for about every 5PDX1-negative definitive endoderm cells can be produced. Additionally,cell cultures or cell populations comprising other ratios ofPDX1-positive foregut endoderm cells to PDX1-negative definitiveendoderm cells are contemplated. For example, compositions comprising atleast about 1 PDX1-positive foregut endoderm cell for about every1,000,000 PDX1-negative definitive endoderm cells, at least about 1PDX1-positive foregut endoderm cell for about every 100,000PDX1-negative definitive endoderm cells, at least about 1 PDX1-positiveforegut endoderm cell for about every 10,000 PDX1-negative definitiveendoderm cells, at least about 1 PDX1-positive foregut endoderm cell forabout every 1000 PDX1-negative definitive endoderm cells, at least about1 PDX1-positive foregut endoderm cell for about every 500 PDX1-negativedefinitive endoderm cells, at least about 1 PDX1-positive foregutendoderm cell for about every 100 PDX1-negative definitive endodermcells, at least about 1 PDX1-positive foregut endoderm cell for aboutevery 10 PDX1-negative definitive endoderm cells, at least about 1PDX1-positive foregut endoderm cell for about every 5 PDX1-negativedefinitive endoderm cells, at least about 1 PDX1-positive foregutendoderm cell for about every 4 PDX1-negative definitive endoderm cells,at least about 1 PDX1-positive foregut endoderm cell for about every 2PDX1-negative definitive endoderm cells, at least about 1 PDX-1 positiveforegut endoderm cell for about every 1 PDX1-negative definitiveendoderm cell, at least about 2 PDX1-positive foregut endoderm cells forabout every 1 PDX1-negative definitive endoderm cell, at least about 4PDX1-positive foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 5 PDX1-positive foregutendoderm cells for about every 1 PDX1-negative definitive endoderm cell,at least about 10 PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 20 PDX1-positiveforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell, at least about 50 PDX1-positive foregut endoderm cellsfor about every 1 PDX1-negative definitive endoderm cell, at least about100 PDX1-positive foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 1000 PDX1-positive foregutendoderm cells for about every 1 PDX1-negative definitive endoderm cell,at least about 10,000 PDX1-positive foregut endoderm cells for aboutevery 1 PDX1-negative definitive endoderm cell, at least about 100,000PDX1-positive foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell and at least about 1,000,000 PDX1-positiveforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell are contemplated.

In some embodiments of the present invention, the PDX1-negativedefinitive endoderm cells from which PDX1-positive foregut endodermcells are produced are derived from human pluripotent cells, such ashuman pluripotent stem cells. In certain embodiments, the humanpluripotent cells are derived from a morula, the inner cell mass of anembryo or the gonadal ridges of an embryo. In certain other embodiments,the human pluripotent cells are derived from the gondal or germ tissuesof a multicellular structure that has developed past the embryonicstage.

Further embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising human cells,including human PDX1-positive foregut endoderm, wherein the expressionof the PDX1 marker is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in at least about 2% of the human cells. Inother embodiments, the expression of the PDX1 marker is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the human cells, in at least about 10% of the human cells,in at least about 15% of the human cells, in at least about 20% of thehuman cells, in at least about 25% of the human cells, in at least about30% of the human cells, in at least about 35% of the human cells, in atleast about 40% of the human cells, in at least about 45% of the humancells, in at least about 50% of the human cells, in at least about 55%of the human cells, in at least about 60% of the human cells, in atleast about 65% of the human cells, in at least about 70% of the humancells, in at least about 75% of the human cells, in at least about 80%of the human cells, in at least about 85% of the human cells, in atleast about 90% of the human cells, in at least about 95% of the humancells or in at least about 98% of the human cells. In some embodiments,the percentage of human cells in the cell cultures or populations,wherein the expression of PDX1 is greater than the expression of theAFP, SOX7, SOX1, ZIC1 and/or NFM marker, is calculated without regard tofeeder cells.

It will be appreciated that some embodiments of the present inventionrelate to compositions, such as cell cultures or cell populations,comprising human PDX1-positive foregut endoderm cells, wherein theexpression of one or more markers selected from the group consisting ofSOX17, HOXA13 and HOXC6 is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in from at least about 2% to greater thanat least about 98% of the human cells. In some embodiments, theexpression of one or more markers selected from the group consisting ofSOX17, HOXA13 and HOXC6 is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in at least about 5% of the human cells, inat least about 10% of the human cells, in at least about 15% of thehuman cells, in at least about 20% of the human cells, in at least about25% of the human cells, in at least about 30% of the human cells, in atleast about 35% of the human cells, in at least about 40% of the humancells, in at least about 45% of the human cells, in at least about 50%of the human cells, in at least about 55% of the human cells, in atleast about 60% of the human cells, in at least about 65% of the humancells, in at least about 70% of the human cells, in at least about 75%of the human cells, in at least about 80% of the human cells, in atleast about 85% of the human cells, in at least about 90% of the humancells, in at least about 95% of the human cells or in at least about 98%of the human cells. In some embodiments, the percentage of human cellsin the cell cultures or populations, wherein the expression of one ormore markers selected from the group consisting of SOX17, HOXA13 andHOXC6 is greater than the expression of the AFP, SOX7, SOX1, ZIC1 and/orNFM marker, is calculated without regard to feeder cells.

Additional embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mammalianendodermal cells, such as human endoderm cells, wherein the expressionof the PDX1 marker is greater than the expression of the AFP, SOX7,SOX1, ZIC1 and/or NFM marker in at least about 2% of the endodermalcells. In other embodiments, the expression of the PDX1 marker isgreater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFMmarker in at least about 5% of the endodermal cells, in at least about10% of the endodermal cells, in at least about 15% of the endodermalcells, in at least about 20% of the endodermal cells, in at least about25% of the endodermal cells, in at least about 30% of the endodermalcells, in at least about 35% of the endodermal cells, in at least about40% of the endodermal cells, in at least about 45% of the endodermalcells, in at least about 50% of the endodermal cells, in at least about55% of the endodermal cells, in at least about 60% of the endodermalcells, in at least about 65% of the endodermal cells, in at least about70% of the endodermal cells, in at least about 75% of the endodermalcells, in at least about 80% of the endodermal cells, in at least about85% of the endodermal cells, in at least about 90% of the endodermalcells, in at least about 95% of the endodermal cells or in at leastabout 98% of the endodermal cells.

Still other embodiments of the present invention relate to compositions,such as cell cultures or cell populations, comprising mammalianendodermal cells, such as human endodermal cells, wherein the expressionof one or more markers selected from the group consisting of SOX17,HOXA13 and HOXC6 is greater than the expression of the AFP, SOX7, SOX1,ZIC1 and/or NFM marker in at least about 2% of the endodermal cells. Inother embodiments, the expression of one or more markers selected fromthe group consisting of SOX17, HOXA13 and HOXC6 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the endodermal cells, in at least about 10% of theendodermal cells, in at least about 15% of the endodermal cells, in atleast about 20% of the endodermal cells, in at least about 25% of theendodermal cells, in at least about 30% of the endodermal cells, in atleast about 35% of the endodermal cells, in at least about 40% of theendodermal cells, in at least about 45% of the endodermal cells, in atleast about 50% of the endodermal cells, in at least about 55% of theendodermal cells, in at least about 60% of the endodermal cells, in atleast about 65% of the endodermal cells, in at least about 70% of theendodermal cells, in at least about 75% of the endodermal cells, in atleast about 80% of the endodermal cells, in at least about 85% of theendodermal cells, in at least about 90% of the endodermal cells, in atleast about 95% of the endodermal cells or at least about 98% of theendodermal cells.

Using the processes described herein, compositions comprisingPDX1-positive foregut endoderm cells substantially free of other celltypes can be produced. With respect to cells in cell cultures or in cellpopulations, the term “substantially free of” means that the specifiedcell type of which the cell culture or cell population is free, ispresent in an amount of less than about 5% of the total number of cellspresent in the cell culture or cell population. In some embodiments ofthe present invention, the PDX1-positive foregut endoderm cellpopulations or cell cultures produced by the methods described hereinare substantially free of cells that significantly express the AFP,SOX7, SOX1, ZIC1 and/or NFM marker genes.

In one embodiment of the present invention, a description of aPDX1-positive foregut endoderm cell based on the expression of markergenes is, PDX1 high, AFP low, SOX7 low, SOX1 low, ZIC1 low and NFM low.

Increasing Expression of PDX1 in a SOX17-Positive Definitive EndodermCell

Some aspects of the present invention are related to methods ofincreasing the expression of the PDX1 gene product in cell cultures orcell populations comprising SOX17-positive definitive endoderm cells. Insuch embodiments, the SOX17-positive definitive endoderm cells arecontacted with a differentiation factor in an amount that is sufficientto increase the expression of the PDX1 gene product. The SOX17-positivedefinitive endoderm cells that are contacted with the differentiationfactor can be either PDX1-negative or PDX1-positive. In someembodiments, the differentiation factor can be a retinoid. In certainembodiments, SOX17-positive definitive endoderm cells are contacted witha retinoid at a concentration ranging from about 0.01 μM to about 50 μM.In a preferred embodiment, the retinoid is RA.

In other embodiments of the present invention, the expression of thePDX1 gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with a differentiation factor of the fibroblastgrowth factor family Such differentiation factors can either be usedalone or in conjunction with RA. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with a fibroblast growth factorat a concentration ranging from about 10 ng/ml to about 1000 ng/ml. In apreferred embodiment, the FGF growth factor is FGF-10.

In some embodiments of the present invention, the expression of the PDX1gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with B27. This differentiation factor can either beused alone or in conjunction with one or both of retinoid and FGF familydifferentiation factors. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with B27 at a concentrationranging from about 0.1% (v/v) to about 20% (v/v). In a preferredembodiment, the SOX17-positive definitive endoderm cells are contactedwith RA, FGF-10 and B27.

Methods for increasing the expression of the PDX1 gene product in cellcultures or cell populations comprising SOX17-positive definitiveendoderm cells can be carried out in growth medium containing reduced orno serum. In some embodiments, serum concentrations range from about0.05% (v/v) to about 20% (v/v). In some embodiments, the SOX17-positivecells are grown with serum replacement.

Identification of Factors Capable of Promoting the Differentiation ofPDX1-Negative Definitive Endoderm Cells to PDX1-Positive ForegutEndoderm Cells

Additional aspects of the present invention relate to methods ofidentifying one or more differentiation factors capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In such methods, a cell culture orcell population comprising PDX1-negative definitive endoderm cells isobtained and the expression of PDX1 in the cell culture or cellpopulation is determined. After determining the expression of PDX1, thecells of the cell culture or cell population are contacted with acandidate differentiation factor. In some embodiments, the expression ofPDX1 is determined at the time of contacting or shortly after contactingthe cells with a candidate differentiation factor. PDX1 expression isthen determined at one or more times after contacting the cells with thecandidate differentiation factor. If the expression of PDX1 hasincreased after contact with the candidate differentiation factor ascompared to PDX1 expression prior to contact with the candidatedifferentiation factor, the candidate differentiation factor isidentified as capable of promoting the differentiation of PDX1-negativedefinitive endoderm cells to PDX1-positive foregut endoderm cells.

In some embodiments, the above-described methods of identifying factorscapable of promoting the differentiation of PDX1-negative definitiveendoderm cells to PDX1-positive foregut endoderm cells also includedetermining the expression of the HOXA13 gene and/or the HOXC6 gene inthe cell culture or cell population. In such embodiments, the expressionof HOXA13 and/or HOXC6 is determined both before and after the cells arecontacted with the candidate differentiation factor. If the expressionof PDX1 and HOXA13 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXA13 expression priorto contact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. Similarly, if the expression ofPDX1 and HOXC6 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXC6 expression prior tocontact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In a preferred embodiment, acandidate differentiation factor is identified as being capable ofpromoting the differentiation of PDX1-negative definitive endoderm cellsto PDX1-positive foregut endoderm cells by determining the expression ofPDX1, HOXA13 and HOXC6 both before and after contacting the cells of thecell culture or cell population with the candidate differentiationfactor. In preferred embodiments, the expression of PDX1, HOXA13 and/orHOXC6 is determined Q-PCR.

It will be appreciated that in some embodiments, the expression of oneor more of PDX1, HOXA13 and HOXC6 can be determined at the time ofcontacting or shortly after contacting the cells of the cell cultures orcell populations with a candidate differentiation factor rather thanprior to contacting the cells with a candidate differentiation factor.In such embodiments, the expression of one or more of PDX1, HOXA13 andHOXC6 at the time of contacting or shortly after contacting the cellswith a candidate differentiation factor is compared to the expression ofone or more of PDX1, HOXA13 and HOXC6 at one or more times aftercontacting the cells with a candidate differentiation factor.

In some embodiments of the above-described methods, the one or moretimes at which PDX1 expression is determined after contacting the cellswith the candidate differentiation factor can range from about 1 hour toabout 10 days. For example, PDX1 expression can be determined about 1hour after contacting the cells with the candidate differentiationfactor, about 2 hours after contacting the cells with the candidatedifferentiation factor, about 4 hours after contacting the cells withthe candidate differentiation factor, about 6 hours after contacting thecells with the candidate differentiation factor, about 8 hours aftercontacting the cells with the candidate differentiation factor, about 10hours after contacting the cells with the candidate differentiationfactor, about 12 hours after contacting the cells with the candidatedifferentiation factor, about 16 hours after contacting the cells withthe candidate differentiation factor, about 24 hours after contactingthe cells with the candidate differentiation factor, about 2 days aftercontacting the cells with the candidate differentiation factor, about 3days after contacting the cells with the candidate differentiationfactor, about 4 days after contacting the cells with the candidatedifferentiation factor, about 5 days after contacting the cells with thecandidate differentiation factor, about 6 days after contacting thecells with the candidate differentiation factor, about 7 days aftercontacting the cells with the candidate differentiation factor, about 8days after contacting the cells with the candidate differentiationfactor, about 9 days after contacting the cells with the candidatedifferentiation factor, about 10 days after contacting the cells withthe candidate differentiation factor or more than 10 days aftercontacting the cells with the candidate differentiation factor.

Candidate differentiation factors for use in the methods describedherein can be selected from compounds, such as polypeptides and smallmolecules. For example, candidate polypeptides can include, but are notlimited to, growth factors, cytokines, chemokines, extracellular matrixproteins, and synthetic peptides. In a preferred embodiment, the growthfactor is from the FGF family, for example FGF-10. Candidate smallmolecules include, but are not limited to, compounds generated fromcombinatorial chemical synthesis and natural products, such as steroids,isoprenoids, terpenoids, phenylpropanoids, alkaloids and flavinoids. Itwill be appreciated by those of ordinary skill in the art that thousandsof classes of natural and synthetic small molecules are available andthat the small molecules contemplated for use in the methods describedherein are not limited to the classes exemplified above. Typically,small molecules will have a molecular weight less than 10,000 amu. In apreferred embodiment, the small molecule is a retinoid, for example RA.

Identification of Factors Capable of Promoting the Differentiation ofPDX1-Positive Foregut Endoderm Cells

Other aspects of the present invention relate to methods of identifyingone or more differentiation factors capable of promoting thedifferentiation of PDX1-positive foregut endoderm cells. In suchmethods, a cell culture or cell population comprising PDX1-positiveforegut endoderm cells is obtained and the expression of a marker in thecell culture or cell population is determined. After determining theexpression of the marker, the cells of the cell culture or cellpopulation are contacted with a candidate differentiation factor. Insome embodiments, the expression of the marker is determined at the timeof contacting or shortly after contacting the cells with a candidatedifferentiation factor. The expression of the same marker is thendetermined at one or more times after contacting the cells with thecandidate differentiation factor. If the expression of the marker hasincreased or decreased after contact with the candidate differentiationfactor as compared to the marker expression prior to contact with thecandidate differentiation factor, the candidate differentiation factoris identified as capable of promoting the differentiation ofPDX1-positive foregut endoderm cells. In preferred embodiments,expression of the marker is determined by Q-PCR.

In some embodiments of the above-described methods, the one or moretimes at which the marker expression is determined after contacting thecells with the candidate differentiation factor can range from about 1hour to about 10 days. For example, marker expression can be determinedabout 1 hour after contacting the cells with the candidatedifferentiation factor, about 2 hours after contacting the cells withthe candidate differentiation factor, about 4 hours after contacting thecells with the candidate differentiation factor, about 6 hours aftercontacting the cells with the candidate differentiation factor, about 8hours after contacting the cells with the candidate differentiationfactor, about 10 hours after contacting the cells with the candidatedifferentiation factor, about 12 hours after contacting the cells withthe candidate differentiation factor, about 16 hours after contactingthe cells with the candidate differentiation factor, about 24 hoursafter contacting the cells with the candidate differentiation factor,about 2 days after contacting the cells with the candidatedifferentiation factor, about 3 days after contacting the cells with thecandidate differentiation factor, about 4 days after contacting thecells with the candidate differentiation factor, about 5 days aftercontacting the cells with the candidate differentiation factor, about 6days after contacting the cells with the candidate differentiationfactor, about 7 days after contacting the cells with the candidatedifferentiation factor, about 8 days after contacting the cells with thecandidate differentiation factor, about 9 days after contacting thecells with the candidate differentiation factor, about 10 days aftercontacting the cells with the candidate differentiation factor or morethan 10 days after contacting the cells with the candidatedifferentiation factor.

As described previously, candidate differentiation factors for use inthe methods described herein can be selected from compounds such aspolypeptides and small molecules.

Although each of the methods disclosed herein have been described withrespect to PDX1-positive foregut endoderm cells, it will be appreciatedthat in certain embodiments, these methods can be used to producecompositions comprising the PDX1-positive foregut/midgut endoderm cellsthat are described herein and/or the PDX1-positive endoderm cells of theposterior portion of the foregut that are described herein. Furthermore,any of the PDX1-positive endoderm cell types disclosed in thisspecification can be utilized in the screening methods described herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting.

EXAMPLES

Many of the examples below describe the use of pluripotent human cells.Methods of producing pluripotent human cells are well known in the artand have been described numerous scientific publications, including U.S.Pat. Nos. 5,453,357, 5,670,372, 5,690,926, 6,090,622, 6,200,806 and6,251,671 as well as U.S. Patent Application Publication No.2004/0229350, the disclosures of which are incorporated herein byreference in their entireties.

Example 1 Human ES Cells

For our studies of endoderm development we employed human embryonic stemcells, which are pluripotent and can divide seemingly indefinitely inculture while maintaining a normal karyotype. ES cells were derived fromthe 5-day-old embryo inner cell mass using either immunological ormechanical methods for isolation. In particular, the human embryonicstem cell line hESCyt-25 was derived from a supernumerary frozen embryofrom an in vitro fertilization cycle following informed consent by thepatient. Upon thawing the hatched blastocyst was plated on mouseembryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non essentialamino acids, beta-mercaptoethanol, ITS supplement). The embryo adheredto the culture dish and after approximately two weeks, regions ofundifferentiated hESCs were transferred to new dishes with MEFs.Transfer was accomplished with mechanical cutting and a brief digestionwith dispase, followed by mechanical removal of the cell clusters,washing and re-plating. Since derivation, hESCyt-25 has been seriallypassaged over 100 times. We employed the hESCyt-25 human embryonic stemcell line as our starting material for the production of definitiveendoderm.

It will be appreciated by those of skill in the art that stem cells orother pluripotent cells can also be used as starting material for thedifferentiation procedures described herein. For example, cells obtainedfrom embryonic gonadal ridges, which can be isolated by methods known inthe art, can be used as pluripotent cellular starting material.

Example 2 hESCyt-25 Characterization

The human embryonic stem cell line, hESCyt-25 has maintained a normalmorphology, karyotype, growth and self-renewal properties over 18 monthsin culture. This cell line displays strong immunoreactivity for theOCT4, SSEA-4 and TRA-1-60 antigens, all of which, are characteristic ofundifferentiated hESCs and displays alkaline phosphatase activity aswell as a morphology identical to other established hESC lines.Furthermore, the human stem cell line, hESCyt-25, also readily formsembryoid bodies (EBs) when cultured in suspension. As a demonstration ofits pluripotent nature, hESCyT-25 differentiates into various cell typesthat represent the three principal germ layers. Ectoderm production wasdemonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC) fornestin and more mature neuronal markers. Immunocytochemical staining forβ-III tubulin was observed in clusters of elongated cells,characteristic of early neurons. Previously, we treated EBs insuspension with retinoic acid, to induce differentiation of pluripotentstem cells to visceral endoderm (VE), an extra-embryonic lineage.Treated cells expressed high levels of α-fetoprotein (AFP) and SOX7, twomarkers of VE, by 54 hours of treatment. Cells differentiated inmonolayer expressed AFP in sporadic patches as demonstrated byimmunocytochemical staining. As will be described below, the hESCyT-25cell line was also capable of forming definitive endoderm, as validatedby real-time quantitative polymerase chain reaction (Q-PCR) andimmunocytochemistry for SOX17, in the absence of AFP expression. Todemonstrate differentiation to mesoderm, differentiating EBs wereanalyzed for Brachyury gene expression at several time points. Brachyuryexpression increased progressively over the course of the experiment. Inview of the foregoing, the hESCyT-25 line is pluripotent as shown by theability to form cells representing the three germ layers.

Example 3 Production of SOX17 Antibody

A primary obstacle to the identification of definitive endoderm in hESCcultures is the lack of appropriate tools. We therefore undertook theproduction of an antibody raised against human SOX17 protein.

The marker SOX17 is expressed throughout the definitive endoderm as itforms during gastrulation and its expression is maintained in the guttube (although levels of expression vary along the A-P axis) untilaround the onset of organogenesis. SOX17 is also expressed in a subsetof extra-embryonic endoderm cells. No expression of this protein hasbeen observed in mesoderm or ectoderm. It has now been discovered thatSOX17 is an appropriate marker for the definitive endoderm lineage whenused in conjunction with markers to exclude extra-embryonic lineages.

As described in detail herein, the SOX17 antibody was utilized tospecifically examine effects of various treatments and differentiationprocedures aimed at the production of SOX17 positive definitive endodermcells. Other antibodies reactive to AFP, SPARC and Thrombomodulin werealso employed to rule out the production of visceral and parietalendoderm (extra-embryonic endoderm).

In order to produce an antibody against SOX17, a portion of the humanSOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids 172-414 (SEQ IDNO: 2) in the carboxyterminal end of the SOX17 protein (FIG. 2) was usedfor genetic immunization in rats at the antibody production company,GENOVAC (Freiberg, Germany), according to procedures developed there.Procedures for genetic immunization can be found in U.S. Pat. Nos.5,830,876, 5,817,637, 6,165,993 and 6,261,281 as well as InternationalPatent Application Publication Nos. WO00/29442 and WO99/13915, thedisclosures of which are incorporated herein by reference in theirentireties.

Other suitable methods for genetic immunization are also described inthe non-patent literature. For example, Barry et al. describe theproduction of monoclonal antibodies by genetic immunization inBiotechniques 16: 616-620, 1994, the disclosure of which is incorporatedherein by reference in its entirety. Specific examples of geneticimmunization methods to produce antibodies against specific proteins canbe found, for example, in Costaglia et al., (1998) Genetic immunizationagainst the human thyrotropin receptor causes thyroiditis and allowsproduction of monoclonal antibodies recognizing the native receptor, J.Immunol. 160: 1458-1465; Kilpatrick et al (1998) Gene gun deliveredDNA-based immunizations mediate rapid production of murine monoclonalantibodies to the Flt-3 receptor, Hybridoma 17: 569-576; Schmolke etal., (1998) Identification of hepatitis G virus particles in human serumby E2-specific monoclonal antibodies generated by DNA immunization, J.Virol. 72: 4541-4545; Krasemann et al., (1999) Generation of monoclonalantibodies against proteins with an unconventional nucleic acid-basedimmunization strategy, J. Biotechnol. 73: 119-129; and Ulivieri et al.,(1996) Generation of a monoclonal antibody to a defined portion of theHeliobacter pylori vacuolating cytotoxin by DNA immunization, J.Biotechnol. 51: 191-194, the disclosures of which are incorporatedherein by reference in their entireties.

SOX7 and SOX18 are the closest Sox family relatives to SOX17 as depictedin the relational dendrogram shown in FIG. 3. We employed the human SOX7polypeptide as a negative control to demonstrate that the SOX17 antibodyproduced by genetic immunization is specific for SOX17 and does notreact with its closest family member. In particular, SOX7 and otherproteins were expressed in human fibroblasts, and then, analyzed forcross reactivity with the SOX17 antibody by Western blot and ICC. Forexample, the following methods were utilized for the production of theSOX17, SOX7 and EGFP expression vectors, their transfection into humanfibroblasts and analysis by Western blot. Expression vectors employedfor the production of SOX17, SOX7, and EGFP were pCMV6 (OriGeneTechnologies, Inc., Rockville, Md.), pCMV-SPORT6 (Invitrogen, Carlsbad,Calif.) and pEGFP-N1 (Clonetech, Palo Alto, Calif.), respectively. Forprotein production, telomerase immortalized MDX human fibroblasts weretransiently transfected with supercoiled DNA in the presence ofLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Total cellularlysates were collected 36 hours post-transfection in 50 mM TRIS-HCl (pH8), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail ofprotease inhibitors (Roche Diagnostics Corporation, Indianapolis, Ind.).Western blot analysis of 100 μg of cellular proteins, separated bySDS-PAGE on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad,Calif.), and transferred by electro-blotting onto PDVF membranes(Hercules, Calif.), were probed with a 1/1000 dilution of the rat SOX17anti-serum in 10 mM TRIS-HCl (pH 8), 150 mM NaCl, 10% BSA, 0.05%Tween-20 (Sigma, St. Louis, Mo.), followed by Alkaline Phosphataseconjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, WestGrove, Pa.), and revealed through Vector Black Alkaline Phosphatasestaining (Vector Laboratories, Burlingame, Calif.). The proteins sizestandard used was wide range color markers (Sigma, St. Louis, Mo.).

In FIG. 4, protein extracts made from human fibroblast cells that weretransiently transfected with SOX17, SOX7 or EGFP cDNA's were probed onWestern blots with the SOX17 antibody. Only the protein extract fromhSOX17 transfected cells produced a band of ˜51Kda which closely matchedthe predicted 46 Kda molecular weight of the human SOX17 protein. Therewas no reactivity of the SOX17 antibody to extracts made from eitherhuman SOX7 or EGFP transfected cells. Furthermore, the SOX17 antibodyclearly labeled the nuclei of human fibroblast cells transfected withthe hSOX17 expression construct but did not label cells transfected withEGFP alone. As such, the SOX17 antibody exhibits specificity by ICC.

Example 4 Validation of SOX17 Antibody as a Marker of DefinitiveEndoderm

Partially differentiated hESCs were co-labeled with SOX17 and AFPantibodies to demonstrate that the SOX17 antibody is specific for humanSOX17 protein and furthermore marks definitive endoderm. It has beendemonstrated that SOX17, SOX7 (which is a closely related member of theSOX gene family subgroup F (FIG. 3)) and AFP are each expressed invisceral endoderm. However, AFP and SOX7 are not expressed in definitiveendoderm cells at levels detectable by ICC, and thus, they can beemployed as negative markers for bonifide definitive endoderm cells. Itwas shown that SOX17 antibody labels populations of cells that exist asdiscrete groupings of cells or are intermingled with AFP positive cells.In particular, FIG. 5A shows that small numbers of SOX17 cells wereco-labeled with AFP; however, regions were also found where there werelittle or no AFP⁺ cells in the field of SOX17⁺ cells (FIG. 5B).Similarly, since parietal endoderm has been reported to express SOX17,antibody co-labeling with SOX17 together with the parietal markers SPARCand/or Thrombomodulin (TM) can be used to identify the SOX17⁺ cells thatare parietal endoderm. As shown in FIGS. 6A-6C, Thrombomodulin and SOX17co-labeled parietal endoderm cells were produced by randomdifferentiation of hES cells.

In view of the above cell labeling experiments, the identity of adefinitive endoderm cell can be established by the marker profileSOX17^(hi)/AFP^(lo)/[TM^(lo) or SPARC^(lo)]. In other words, theexpression of the SOX17 marker is greater than the expression of the AFPmarker, which is characteristic of visceral endoderm, and the TM orSPARC markers, which are characteristic of parietal endoderm.Accordingly, those cells positive for SOX17 but negative for AFP andnegative for TM or SPARC are definitive endoderm.

As a further evidence of the specificity of theSOX17^(hi)/AFP^(lo)/TM^(lo)/SPARC^(lo) marker profile as predictive ofdefinitive endoderm, SOX17 and AFP gene expression was quantitativelycompared to the relative number of antibody labeled cells. As shown inFIG. 7A, hESCs treated with retinoic acid (visceral endoderm inducer),or activin A (definitive endoderm inducer), resulted in a 10-folddifference in the level of SOX17 mRNA expression. This result mirroredthe 10-fold difference in SOX17 antibody-labeled cell number (FIG. 7B).Furthermore, as shown in FIG. 8A, activin A treatment of hESCssuppressed AFP gene expression by 6.8-fold in comparison to notreatment. This was visually reflected by a dramatic decrease in thenumber of AFP labeled cells in these cultures as shown in FIGS. 8B-8C.To quantify this further, it was demonstrated that this approximately7-fold decrease in AFP gene expression was the result of a similar7-fold decrease in AFP antibody-labeled cell number as measured by flowcytometry (FIGS. 9A-9B). This result is extremely significant in that itindicates that quantitative changes in gene expression as seen by Q-PCRmirror changes in cell type specification as observed by antibodystaining.

Incubation of hESCs in the presence of Nodal family members (Nodal,activin A and activin B—NAA) resulted in a significant increase in SOX17antibody-labeled cells over time. By 5 days of continuous activintreatment greater than 50% of the cells were labeled with SOX17 (FIGS.10A-10F). There were few or no cells labeled with AFP after 5 days ofactivin treatment.

In summary, the antibody produced against the carboxy-terminal 242 aminoacids of the human SOX17 protein identified human SOX17 protein onWestern blots but did not recognize SOX7, it's closest Sox familyrelative. The SOX17 antibody recognized a subset of cells indifferentiating hESC cultures that were primarily SOX17⁺/AFP^(lo/-)(greater than 95% of labeled cells) as well as a small percentage (<5%)of cells that co-label for SOX17 and AFP (visceral endoderm). Treatmentof hESC cultures with activins resulted in a marked elevation of SOX17gene expression as well as SOX17 labeled cells and dramaticallysuppressed the expression of AFP mRNA and the number of cells labeledwith AFP antibody.

Example 5 Q-PCR Gene Expression Assay

In the following experiments, real-time quantitative RT-PCR (Q-PCR) wasthe primary assay used for screening the effects of various treatmentson hESC differentiation. In particular, real-time measurements of geneexpression were analyzed for multiple marker genes at multiple timepoints by Q-PCR. Marker genes characteristic of the desired as well asundesired cell types were evaluated to gain a better understanding ofthe overall dynamics of the cellular populations. The strength of Q-PCRanalysis includes its extreme sensitivity and relative ease ofdeveloping the necessary markers, as the genome sequence is readilyavailable. Furthermore, the extremely high sensitivity of Q-PCR permitsdetection of gene expression from a relatively small number of cellswithin a much larger population. In addition, the ability to detect verylow levels of gene expression provides indications for “differentiationbias” within the population. The bias towards a particulardifferentiation pathway, prior to the overt differentiation of thosecellular phenotypes, is unrecognizable using immunocytochemicaltechniques. For this reason, Q-PCR provides a method of analysis that isat least complementary and potentially much superior toimmunocytochemical techniques for screening the success ofdifferentiation treatments. Additionally, Q-PCR provides a mechanism bywhich to evaluate the success of a differentiation protocol in aquantitative format at semi-high throughput scales of analysis.

The approach taken here was to perform relative quantitation using SYBRGreen chemistry on a Rotor Gene 3000 instrument (Corbett Research) and atwo-step RT-PCR format. Such an approach allowed for the banking of cDNAsamples for analysis of additional marker genes in the future, thusavoiding variability in the reverse transcription efficiency betweensamples.

Primers were designed to lie over exon-exon boundaries or span intronsof at least 800 bp when possible, as this has been empiricallydetermined to eliminate amplification from contaminating genomic DNA.When marker genes were employed that do not contain introns or theypossess pseudogenes, DNase I treatment of RNA samples was performed.

We routinely used Q-PCR to measure the gene expression of multiplemarkers of target and non-target cell types in order to provide a broadprofile description of gene expression in cell samples. The markersrelevant for the early phases of hESC differentiation (specificallyectoderm, mesoderm, definitive endoderm and extra-embryonic endoderm)and for which validated primer sets are available are provided below inTable 1. The human specificity of these primer sets has also beendemonstrated. This is an important fact since the hESCs were often grownon mouse feeder layers. Most typically, triplicate samples were takenfor each condition and independently analyzed in duplicate to assess thebiological variability associated with each quantitative determination.

To generate PCR template, total RNA was isolated using RNeasy (Qiagen)and quantitated using RiboGreen (Molecular Probes). Reversetranscription from 350-500 ng of total RNA was carried out using theiScript reverse transcriptase kit (BioRad), which contains a mix ofoligo-dT and random primers. Each 20 μL reaction was subsequentlydiluted up to 100 μL total volume and 3 μL was used in each 10 μL Q-PCRreaction containing 400 nM forward and reverse primers and 5 μL 2×SYBRGreen master mix (Qiagen). Two step cycling parameters were usedemploying a 5 second denature at 85-94° C. (specifically selectedaccording to the melting temp of the amplicon for each primer set)followed by a 45 second anneal/extend at 60° C. Fluorescence data wascollected during the last 15 seconds of each extension phase. A threepoint, 10-fold dilution series was used to generate the standard curvefor each run and cycle thresholds (Ct's) were converted to quantitativevalues based on this standard curve. The quantitated values for eachsample were normalized to housekeeping gene performance and then averageand standard deviations were calculated for triplicate samples. At theconclusion of PCR cycling, a melt curve analysis was performed toascertain the specificity of the reaction. A single specific product wasindicated by a single peak at the T_(m) appropriate for that PCRamplicon. In addition, reactions performed without reverse transcriptaseserved as the negative control and do not amplify.

A first step in establishing the Q-PCR methodology was validation ofappropriate housekeeping genes (HGs) in the experimental system. Sincethe HG was used to normalize across samples for the RNA input, RNAintegrity and RT efficiency, it was of value that the HG exhibited aconstant level of expression over time in all sample types in order forthe normalization to be meaningful. We measured the expression levels ofCyclophilin G, hypoxanthine phosphoribosyltransferase 1 (HPRT),beta-2-microglobulin, hydroxymethylbiane synthase (HMBS), TATA-bindingprotein (TBP), and glucoronidase beta (GUS) in differentiating hESCs.Our results indicated that beta-2-microglobulin expression levelsincreased over the course of differentiation and therefore we excludedthe use of this gene for normalization. The other genes exhibitedconsistent expression levels over time as well as across treatments. Weroutinely used both Cyclophilin G and GUS to calculate a normalizationfactor for all samples. The use of multiple HGs simultaneously reducesthe variability inherent to the normalization process and increases thereliability of the relative gene expression values.

After obtaining genes for use in normalization, Q-PCR was then utilizedto determine the relative gene expression levels of many marker genesacross samples receiving different experimental treatments. The markergenes employed have been chosen because they exhibit enrichment inspecific populations representative of the early germ layers and inparticular have focused on sets of genes that are differentiallyexpressed in definitive endoderm and extra-embryonic endoderm. Thesegenes as well as their relative enrichment profiles are highlighted inTable 1.

TABLE 1 Germ Layer Gene Expression Domains Endoderm SOX17 definitive,visceral and parietal endoderm MIXL1 endoderm and mesoderm GATA4definitive and primitive endoderm HNF3b definitive endoderm andprimitive endoderm, mesoderm, neural plate GSC endoderm and mesodermExtra- SOX7 visceral endoderm embryonic AFP visceral endoderm, liverSPARC parietal endoderm TM parietal endoderm/trophectoderm Ectoderm ZIC1neural tube, neural progenitors Mesoderm BRACH nascent mesoderm

Since many genes are expressed in more than one germ layer it is usefulto quantitatively compare expression levels of many genes within thesame experiment. SOX17 is expressed in definitive endoderm and to asmaller extent in visceral and parietal endoderm. SOX7 and AFP areexpressed in visceral endoderm at this early developmental time point.SPARC and TM are expressed in parietal endoderm and Brachyury isexpressed in early mesoderm.

Definitive endoderm cells were predicted to express high levels of SOX17mRNA and low levels of AFP and SOX7 (visceral endoderm), SPARC (parietalendoderm) and Brachyury (mesoderm). In addition, ZIC1 was used here tofurther rule out induction of early ectoderm. Finally, GATA4 and HNF3bwere expressed in both definitive and extra-embryonic endoderm, andthus, correlate with SOX17 expression in definitive endoderm (Table 1).A representative experiment is shown in FIGS. 11-14 which demonstrateshow the marker genes described in Table 1 correlate with each otheramong the various samples, thus highlighting specific patterns ofdifferentiation to definitive endoderm and extra-embryonic endoderm aswell as to mesodermal and neural cell types.

In view of the above data it is clear that increasing doses of activinresulted in increasing SOX17 gene expression. Further this SOX17expression predominantly represented definitive endoderm as opposed toextra-embryonic endoderm. This conclusion stems from the observationthat SOX17 gene expression was inversely correlated with AFP, SOX7, andSPARC gene expression.

Example 6 Directed Differentiation of Human ES Cells to DefinitiveEndoderm

Human ES cell cultures randomly differentiate if cultured underconditions that do not actively maintain their undifferentiated state.This heterogeneous differentiation results in production ofextra-embryonic endoderm cells comprised of both parietal and visceralendoderm (AFP, SPARC and SOX7 expression) as well as early ectodermaland mesodermal derivatives as marked by ZIC1 and Nestin (ectoderm) andBrachyury (mesoderm) expression. Definitive endoderm cell appearance hasnot been examined or specified for lack of specific antibody markers inES cell cultures. As such, and by default, early definitive endodermproduction in ES cell cultures has not been well studied. Sincesatisfactory antibody reagents for definitive endoderm cells have beenunavailable, most of the characterization has focused on ectoderm andextra-embryonic endoderm. Overall, there are significantly greaternumbers of extra-embryonic and neurectodermal cell types in comparisonto SOX17′ definitive endoderm cells in randomly differentiated ES cellcultures.

As undifferentiated hESC colonies expand on a bed of fibroblast feeders,the cells at the edges of the colony take on alternative morphologiesthat are distinct from those cells residing within the interior of thecolony. Many of these outer edge cells can be distinguished by theirless uniform, larger cell body morphology and by the expression ofhigher levels of OCT4. It has been described that as ES cells begin todifferentiate they alter the levels of OCT4 expression up or downrelative to undifferentiated ES cells. Alteration of OCT4 levels aboveor below the undifferentiated threshold may signify the initial stagesof differentiation away from the pluripotent state.

When undifferentiated colonies were examined by SOX17immunocytochemistry, occasionally small 10-15-cell clusters ofSOX17-positive cells were detected at random locations on the peripheryand at the junctions between undifferentiated hESC colonies. As notedabove, these scattered pockets of outer colony edges appeared to be someof the first cells to differentiate away from the classical ES cellmorphology as the colony expanded in size and became more crowded.Younger, smaller fully undifferentiated colonies (<1 mm; 4-5 days old)showed no SOX17 positive cells within or at the edges of the colonieswhile older, larger colonies (1-2 mm diameter, >5 days old) had sporadicisolated patches of SOX17 positive, AFP negative cells at the peripheryof some colonies or in regions interior to the edge that did not displaythe classical hESC morphology described previously. Given that this wasthe first development of an effective SOX17 antibody, definitiveendoderm cells generated in such early “undifferentiated” ES cellcultures have never been previously demonstrated.

Based on negative correlations of SOX17 and SPARC gene expression levelsby Q-PCR, the vast majority of these SOX17 positive, AFP negative cellswill be negative for parietal endoderm markers by antibody co-labeling.This was specifically demonstrated for TM-expressing parietal endodermcells as shown in FIGS. 15A-15B. Exposure to Nodal factors activin A andB resulted in a dramatic decrease in the intensity of TM expression andthe number of TM positive cells. By triple labeling using SOX17, AFP andTM antibodies on an activin treated culture, clusters of SOX17 positivecells that were also negative for AFP and TM were observed (FIGS.16A-16D). These are the first cellular demonstrations of SOX17 positivedefinitive endoderm cells in differentiating hESC cultures (FIGS.16A-16D and 17).

With the SOX17 antibody and Q-PCR tools described above we have exploreda number of procedures capable of efficiently programming hESCs tobecome SOX17^(hi)/AFP^(lo)/SPARC/TM^(lo) definitive endoderm cells. Weapplied a variety of differentiation protocols aimed at increasing thenumber and proliferative capacity of these cells as measured at thepopulation level by Q-PCR for SOX17 gene expression and at the level ofindividual cells by antibody labeling of SOX17 protein.

We were the first to analyze and describe the effect of TGFβ familygrowth factors, such as Nodal/activin/BMP, for use in creatingdefinitive endoderm cells from embryonic stem cells in in vitro cellcultures. In typical experiments, activin A, activin B, BMP orcombinations of these growth factors were added to cultures ofundifferentiated human stem cell line hESCyt-25 to begin thedifferentiation process.

As shown in FIG. 19, addition of activin A at 100 ng/ml resulted in a19-fold induction of SOX17 gene expression vs. undifferentiated hESCs byday 4 of differentiation. Adding activin B, a second member of theactivin family, together with activin A, resulted in a 37-fold inductionover undifferentiated hESCs by day 4 of combined activin treatment.Finally, adding a third member of the TGFβ family from the Nodal/Activinand BMP subgroups, BMP4, together with activin A and activin B,increased the fold induction to 57 times that of undifferentiated hESCs(FIG. 19). When SOX17 induction with activins and BMP was compared to nofactor medium controls 5-, 10-, and 15-fold inductions resulted at the4-day time point. By five days of triple treatment with activins A, Band BMP, SOX17 was induced more than 70 times higher than hESCs. Thesedata indicate that higher doses and longer treatment times of theNodal/activin TGFβ family members results in increased expression ofSOX17.

Nodal and related molecules activin A, B and BMP facilitate theexpression of SOX17 and definitive endoderm formation in vivo or invitro. Furthermore, addition of BMP results in an improved SOX17induction possibly through the further induction of Cripto, the Nodalco-receptor.

We have demonstrated that the combination of activins A and B togetherwith BMP4 result in additive increases in SOX17 induction and hencedefinitive endoderm formation. BMP4 addition for prolonged periods (>4days), in combination with activin A and B may induce SOX17 in parietaland visceral endoderm as well as definitive endoderm. In someembodiments of the present invention, it is therefore valuable to removeBMP4 from the treatment within 4 days of addition.

To determine the effect of TGFβ factor treatment at the individual celllevel, a time course of TGFβ factor addition was examined using SOX17antibody labeling. As previously shown in FIGS. 10A-10F, there was adramatic increase in the relative number of SOX17 labeled cells overtime. The relative quantification (FIG. 20) shows more than a 20-foldincrease in SOX17-labeled cells. This result indicates that both thenumbers of cells as well SOX17 gene expression level are increasing withtime of TGFβ factor exposure. As shown in FIG. 21, after four days ofexposure to Nodal, activin A, activin B and BMP4, the level of SOX17induction reached 168-fold over undifferentiated hESCs. FIG. 22 showsthat the relative number of SOX17-positive cells was also doseresponsive. activin A doses of 100 ng/ml or more were capable ofpotently inducing SOX17 gene expression and cell number.

In addition to the TGFβ family members, the Wnt family of molecules mayplay a role in specification and/or maintenance of definitive endoderm.The use of Wnt molecules was also beneficial for the differentiation ofhESCs to definitive endoderm as indicated by the increased SOX17 geneexpression in samples that were treated with activins plus Wnt3a overthat of activins alone (FIG. 23).

All of the experiments described above were performed using a tissueculture medium containing 10% serum with added factors. Surprisingly, wediscovered that the concentration of serum had an effect on the level ofSOX17 expression in the presence of added activins as shown in FIGS.24A-24C. When serum levels were reduced from 10% to 2%, SOX17 expressiontripled in the presence of activins A and B.

Finally, we demonstrated that activin induced SOX17⁺ cells divide inculture as depicted in FIGS. 25A-25D. The arrows show cells labeled withSOX17/PCNA/DAPI that are in mitosis as evidenced by thePCNA/DAPI-labeled mitotic plate pattern and the phase contrast mitoticprofile.

Example 7 Chemokine Receptor 4 (CXCR4) Expression Correlates withMarkers for Definitive Endoderm and not Markers for Mesoderm, Ectodermor Visceral Endoderm

As described above, hESCs can be induced to differentiate to thedefinitive endoderm germ layer by the application of cytokines of theTGFβ family and more specifically of the activin/nodal subfamilyAdditionally, we have shown that the proportion of fetal bovine serum(FBS) in the differentiation culture medium effects the efficiency ofdefinitive endoderm differentiation from hESCs. This effect is such thatat a given concentration of activin A in the medium, higher levels ofFBS will inhibit maximal differentiation to definitive endoderm. In theabsence of exogenous activin A, differentiation of hESCs to thedefinitive endoderm lineage is very inefficient and the FBSconcentration has much milder effects on the differentiation process ofhESCs.

In these experiments, hESCs were differentiated by growing in RPMImedium (Invitrogen, Carlsbad, Calif.; cat#61870-036) supplemented with0.5%, 2.0% or 10% FBS and either with or without 100 ng/ml activin A for6 days. In addition, a gradient of FBS ranging from 0.5% to 2.0% overthe first three days of differentiation was also used in conjunctionwith 100 ng/ml of activin A. After the 6 days, replicate samples werecollected from each culture condition and analyzed for relative geneexpression by real-time quantitative PCR. The remaining cells were fixedfor immunofluorescent detection of SOX17 protein.

The expression levels of CXCR4 varied dramatically across the 7 cultureconditions used (FIG. 26). In general, CXCR4 expression was high inactivin A treated cultures (A100) and low in those which did not receiveexogenous activin A (NF). In addition, among the A100 treated cultures,CXCR4 expression was highest when FBS concentration was lowest. Therewas a remarkable decrease in CXCR4 level in the 10% FBS condition suchthat the relative expression was more in line with the conditions thatdid not receive activin A (NF).

As described above, expression of the SOX17, GSC, MIXL1, and HNF313genes is consistent with the characterization of a cell as definitiveendoderm. The relative expression of these four genes across the 7differentiation conditions mirrors that of CXCR4 (FIGS. 27A-27D). Thisdemonstrates that CXCR4 is also a marker of definitive endoderm.

Ectoderm and mesoderm lineages can be distinguished from definitiveendoderm by their expression of various markers. Early mesodermexpresses the genes Brachyury and MOX1 while nascent neuro-ectodermexpresses SOX1 and ZIC1. FIGS. 28A-28D demonstrate that the cultureswhich did not receive exogenous activin A were preferentially enrichedfor mesoderm and ectoderm gene expression and that among the activin Atreated cultures, the 10% FBS condition also had increased levels ofmesoderm and ectoderm marker expression. These patterns of expressionwere inverse to that of CXCR4 and indicated that CXCR4 was not highlyexpressed in mesoderm or ectoderm derived from hESCs at thisdevelopmental time period.

Early during mammalian development, differentiation to extra-embryoniclineages also occurs. Of particular relevance here is thedifferentiation of visceral endoderm that shares the expression of manygenes in common with definitive endoderm, including SOX17. Todistinguish definitive endoderm from extra-embryonic visceral endodermone should examine a marker that is distinct between these two. SOX7represents a marker that is expressed in the visceral endoderm but notin the definitive endoderm lineage. Thus, culture conditions thatexhibit robust SOX17 gene expression in the absence of SOX7 expressionare likely to contain definitive and not visceral endoderm. It is shownin FIG. 28E that SOX7 was highly expressed in cultures that did notreceive activin A, SOX7 also exhibited increased expression even in thepresence of activin A when FBS was included at 10%. This pattern is theinverse of the CXCR4 expression pattern and suggests that CXCR4 is nothighly expressed in visceral endoderm.

The relative number of SOX17 immunoreactive (SOX17⁺) cells present ineach of the differentiation conditions mentioned above was alsodetermined. When hESCs were differentiated in the presence of high doseactivin A and low FBS concentration (0.5%-2.0%) SOX17⁺ cells wereubiquitously distributed throughout the culture. When high dose activinA was used but FBS was included at 10% (v/v), the SOX17⁺ cells appearedat much lower frequency and always appeared in isolated clusters ratherthan evenly distributed throughout the culture (FIGS. 29A and 29C aswell as 29B and 29E). A further decrease in SOX17⁺ cells was seen whenno exogenous activin A was used. Under these conditions the SOX17⁺ cellsalso appeared in clusters and these clusters were smaller and much morerare than those found in the high activin A, low FBS treatment (FIGS.29C and 29F). These results demonstrate that the CXCR4 expressionpatterns not only correspond to definitive endoderm gene expression butalso to the number of definitive endoderm cells in each condition.

Example 8 Differentiation Conditions that Enrich for Definitive EndodermIncrease the Proportion of CXCR4 Positive Cells

The dose of activin A also effects the efficiency at which definitiveendoderm can be derived from hESCs. This example demonstrates thatincreasing the dose of activin A increases the proportion of CXCR4⁺cells in the culture.

hESCs were differentiated in RPMI media supplemented with 0.5%-2% FBS(increased from 0.5% to 1.0% to 2.0% over the first 3 days ofdifferentiation) and either 0, 10, or 100 ng/ml of activin A. After 7days of differentiation the cells were dissociated in PBS withoutCa²⁺/Mg²⁺ containing 2% FBS and 2 mM (EDTA) for 5 minutes at roomtemperature. The cells were filtered through 35 μm nylon filters,counted and pelleted. Pellets were resuspended in a small volume of 50%human serum/50% normal donkey serum and incubated for 2 minutes on iceto block non-specific antibody binding sites. To this, 1 μl of mouseanti-CXCR4 antibody (Abcam, cat# ab10403-100) was added per 50 μl(containing approximately 10⁵ cells) and labeling proceeded for 45minutes on ice. Cells were washed by adding 5 ml of PBS containing 2%human serum (buffer) and pelleted. A second wash with 5 ml of buffer wascompleted then cells were resuspended in 50 μl buffer per 10⁵ cells.Secondary antibody (FITC conjugated donkey anti-mouse; JacksonImmunoResearch, cat#715-096-151) was added at 5 μg/ml finalconcentration and allowed to label for 30 minutes followed by two washesin buffer as above. Cells were resuspended at 5×10⁶ cells/ml in bufferand analyzed and sorted using a FACS Vantage (Beckton Dickenson) by thestaff at the flow cytometry core facility (The Scripps ResearchInstitute). Cells were collected directly into RLT lysis buffer (Qiagen)for subsequent isolation of total RNA for gene expression analysis byreal-time quantitative PCR.

The number of CXCR4⁺ cells as determined by flow cytometry were observedto increase dramatically as the dose of activin A was increased in thedifferentiation culture media (FIGS. 30A-30C). The CXCR4⁺ cells werethose falling within the R4 gate and this gate was set using a secondaryantibody-only control for which 0.2% of events were located in the R4gate. The dramatically increased numbers of CXCR4⁺ cells correlates witha robust increase in definitive endoderm gene expression as activin Adose is increased (FIGS. 31A-31D).

Example 9 Isolation of CXCR4 Positive Cells Enriches for DefinitiveEndoderm Gene Expression and Depletes Cells Expressing Markers ofMesoderm, Ectoderm and Visceral Endoderm

The CXCR4⁺ and CXCR4⁻ cells identified in Example 8 above were collectedand analyzed for relative gene expression and the gene expression of theparent populations was determined simultaneously.

The relative levels of CXCR4 gene expression was dramatically increasedwith increasing dose of activin A (FIG. 32). This correlated very wellwith the activin A dose-dependent increase of CXCR4⁺ cells (FIGS.30A-30C). It is also clear that isolation of the CXCR4⁺ cells from eachpopulation accounted for nearly all of the CXCR4 gene expression in thatpopulation. This demonstrates the efficiency of the FACS method forcollecting these cells.

Gene expression analysis revealed that the CXCR4⁺ cells contain not onlythe majority of the CXCR4 gene expression, but they also contained geneexpression for other markers of definitive endoderm. As shown in FIGS.31A-31D, the CXCR4⁺ cells were further enriched over the parent A100population for SOX17, GSC, HNF3B, and MIXL1. In addition, the CXCR4⁻fraction contained very little gene expression for these definitiveendoderm markers. Moreover, the CXCR4⁺ and CXCR4⁻ populations displayedthe inverse pattern of gene expression for markers of mesoderm, ectodermand extra-embryonic endoderm. FIGS. 33A-33D shows that the CXCR4⁺ cellswere depleted for gene expression of Brachyury, MOX1, ZIC1, and SOX7relative to the A100 parent population. This A100 parent population wasalready low in expression of these markers relative to the low dose orno activin A conditions. These results show that the isolation of CXCR4⁺cells from hESCs differentiated in the presence of high activin A yieldsa population that is highly enriched for and substantially puredefinitive endoderm.

Example 10 Quantitation of Definitive Endoderm Cells in a CellPopulation Using CXCR4

To confirm the quantitation of the proportion of definitive endodermcells present in a cell culture or cell population as determinedpreviously herein and as determined in U.S. Provisional PatentApplication No. 60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23,2003, the disclosure of which is incorporated herein by reference in itsentirety, cells expressing CXCR4 and other markers of definitiveendoderm were analyzed by FACS.

Using the methods such as those described in the above Examples, hESCswere differentiated to produce definitive endoderm. In particular, toincrease the yield and purity in differentiating cell cultures, theserum concentration of the medium was controlled as follows: 0.2% FBS onday 1, 1.0% FBS on day 2 and 2.0% FBS on days 3-6. Differentiatedcultures were sorted by FACS using three cell surface epitopes,E-Cadherin, CXCR4, and Thrombomodulin. Sorted cell populations were thenanalyzed by Q-PCR to determine relative expression levels of markers fordefinitive and extraembryonic-endoderm as well as other cell types.CXCR4 sorted cells taken from optimally differentiated cultures resultedin the isolation of definitive endoderm cells that were >98% pure.

Table 2 shows the results of a marker analysis for a definitive endodermculture that was differentiated from hESCs using the methods describedherein.

TABLE 2 Composition of Definitive Endoderm Cultures Percent PercentPercent Percent of Definitive Extraembryonic hES Marker(s) cultureEndoderm endoderm cells SOX17 70-80 100 Thrombomodulin <2 0 75 AFP <1 025 CXCR4 70-80 100 0 ECAD 10 0 100 other (ECAD neg.) 10-20 Total 100 100 100 100

In particular, Table 2 indicates that CXCR4 and SOX17 positive cells(endoderm) comprised from 70%-80% of the cells in the cell culture. Ofthese SOX17-expressing cells, less than 2% expressed TM (parietalendoderm) and less than 1% expressed AFP (visceral endoderm). Aftersubtracting the proportion of TM-positive and AFP-positive cells(combined parietal and visceral endoderm; 3% total) from the proportionof SOX17/CXCR4 positive cells, it can be seen that about 67% to about77% of the cell culture was definitive endoderm. Approximately 10% ofthe cells were positive for E-Cadherin (ECAD), which is a marker forhESCs, and about 10-20% of the cells were of other cell types.

We have discovered that the purity of definitive endoderm in thedifferentiating cell cultures that are obtained prior to FACS separationcan be improved as compared to the above-described low serum procedureby maintaining the FBS concentration at ≤0.5% throughout the 5-6 daydifferentiation procedure. However, maintaining the cell culture at≤0.5% throughout the 5-6 day differentiation procedure also results in areduced number of total definitive endoderm cells that are produced.

Definitive endoderm cells produced by methods described herein have beenmaintained and expanded in culture in the presence of activin forgreater than 50 days without appreciable differentiation. In such cases,SOX17, CXCR4, MIXL1, GATA4, HNF3β expression is maintained over theculture period. Additionally, TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACHwere not detected in these cultures. It is likely that such cells can bemaintained and expanded in culture for substantially longer than 50 dayswithout appreciable differentiation.

Example 11 Additional Marker of Definitive Endoderm Cells

In the following experiment, RNA was isolated from purified definitiveendoderm and human embryonic stem cell populations. Gene expression wasthen analyzed by gene chip analysis of the RNA from each purifiedpopulation. Q-PCR was also performed to further investigate thepotential of genes expressed in definitive endoderm, but not inembryonic stem cells, as a marker for definitive endoderm.

Human embryonic stem cells (hESCs) were maintained in DMEM/F12 mediasupplemented with 20% KnockOut Serum Replacement, 4 ng/ml recombinanthuman basic fibroblast growth factor (bFGF), 0.1 mM 2-mercaptoethanol,L-glutamine, non-essential amino acids and penicillin/streptomycin.hESCs were differentiated to definitive endoderm by culturing for 5 daysin RPMI media supplemented with 100 ng/ml of recombinant human activinA, fetal bovine serum (FBS), and penicillin/streptomycin. Theconcentration of FBS was varied each day as follows: 0.1% (first day),0.2% (second day), 2% (days 3-5).

Cells were isolated by fluorescence activated cell sorting (FACS) inorder to obtain purified populations of hESCs and definitive endodermfor gene expression analysis. Immuno-purification was achieved for hESCsusing SSEA4 antigen (R&D Systems, cat# FAB1435P) and for definitiveendoderm using CXCR4 (R&D Systems, cat# FAB170P). Cells were dissociatedusing trypsin/EDTA (Invitrogen, cat#25300-054), washed in phosphatebuffered saline (PBS) containing 2% human serum and resuspended in 100%human serum on ice for 10 minutes to block non-specific binding.Staining was carried out for 30 minutes on ice by adding 200 μl ofphycoerythrin-conjugated antibody to 5×10⁶ cells in 800 μl human serum.Cells were washed twice with 8 ml of PBS buffer and resuspended in 1 mlof the same. FACS isolation was carried out by the core facility of TheScripps Research Institute using a FACS Vantage (BD Biosciences). Cellswere collected directly into RLT lysis buffer and RNA was isolated byRNeasy according to the manufacturers instructions (Qiagen).

Purified RNA was submitted in duplicate to Expression Analysis (Durham,N.C.) for generation of the expression profile data using the Affymetrixplatform and U133 Plus 2.0 high-density oligonucleotide arrays. Datapresented is a group comparison that identifies genes differentiallyexpressed between the two populations, hESCs and definitive endoderm.Genes that exhibited a robust upward change in expression level overthat found in hESCs were selected as new candidate markers that arehighly characteristic of definitive endoderm. Select genes were assayedby Q-PCR, as described above, to verify the gene expression changesfound on the gene chip and also to investigate the expression pattern ofthese genes during a time course of hESC differentiation.

FIGS. 34A-34M show the gene expression results for certain markers.Results are displayed for cell cultures analyzed 1, 3 and 5 days afterthe addition of 100 ng/ml activin A, CXCR4-expressing definitiveendoderm cells purified at the end of the five day differentiationprocedure (CXDE), and in purified hESCs. A comparison of FIGS. 34C and34G-34M demonstrates that the six marker genes, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1, exhibit an expression pattern that is almostidentical to each other and which is also identical to the pattern ofexpression of CXCR4 and the ratio of SOX17/SOX7. As describedpreviously, SOX17 is expressed in both the definitive endoderm as wellas in the SOX7-expressing extra-embryonic endoderm. Since SOX7 is notexpressed in the definitive endoderm, the ratio of SOX17/SOX7 provides areliable estimate of definitive endoderm contribution to the SOX17expression witnessed in the population as a whole. The similarity ofpanels G-L and M to panel C indicates that FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1 are likely markers of definitive endoderm and that theyare not significantly expressed in extra-embryonic endoderm cells.

It will be appreciated that the Q-PCR results described herein can befurther confirmed by ICC.

Example 12 Retinoic Acid and FGF-10 Induces PDX1 Specifically inDefinitive Endoderm Cultures

The following experiment demonstrates that RA and FGF-10 induces theexpression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, 1 μM RA and 50 ng/ml FGF-10 were added to thecell culture. Forty-eight hours after the RA/FGF-10 addition, theexpression of the PDX1 marker gene and other marker genes not specificto foregut endoderm were quantitated by Q-PCR.

The application of RA to definitive endoderm cells caused a robustincrease in PDX1 gene expression (see FIG. 35) without increasing theexpression of visceral endoderm (SOX7, AFP), neural (SOX1, ZIC1), orneuronal (NFM) gene expression markers (see FIGS. 36A-36F). PDX1 geneexpression was induced to levels approximately 500-fold higher thanobserved in definitive endoderm after 48 hours exposure to 1 μM RA and50 ng/ml FGF-10. Furthermore, these results show that substantial PDX1induction occurred only in cell cultures which had been previouslydifferentiated to definitive endoderm (SOX17) as indicated by the160-fold higher PDX1 expression found in the activin treated cellcultures relative to those cultures that received no activin prior to RAapplication.

Example 13 FGF-10 Provides Additional Increase in PDX1 Expression OverRA Alone

This Example shows that the combination of RA and FGF-10 induces PDX1expression to a greater extent than RA alone.

As in the previous Example, hESCs were cultured with or without activinsfor four days. On day four, the cells were treated with one of thefollowing: 1 μM RA alone; 1 μM RA in combination with either FGF-4 orFGF-10; or 1 μM RA in combination with both FGF-4 and FGF-10. Theexpression of PDX1, SOX7 and NFM were quantitated by Q-PCR ninety sixhours after RA or RA/FGF.

The treatment of hESC cultures with activin followed by retinoic acidinduced a 60-fold increase in PDX1 gene expression. The addition ofFGF-4 to the RA treatment induced slightly more PDX1 (approximately3-fold over RA alone). However, by adding FGF-10 and retinoic acidtogether, the induction of PDX1 was further enhanced 60-fold over RAalone (see FIG. 37A). This very robust PDX1 induction was greater than1400-fold higher than with no activin or RA/FGF treatment.Interestingly, addition of FGF-4 and FGF-10 simultaneously abolished thebeneficial effect of the FGF-10, producing only the modest PDX1 increaseattributed to FGF-4 addition.

Addition of RA/FGF-4 or RA/FGF-10 combinations did not increase theexpression of marker genes not associated with foregut endoderm whencompared to cells not exposed to RA/FGF combinations (see FIGS.37B-37C).

Example 14 Retinoic Acid Dose Affects Anterior-Posterior (A-P) PositionIn Vitro

To determine whether the dose of RA affects A-P position in in vitrocell cultures, the following experiment was performed.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, FGF-10 at 50 ng/ml was added to the culture incombination with RA at 0.04 μM, 0.2 μM or 1.0 μM. The expression of thePDX1 marker gene as well as other markers not specific for foregutendoderm were quantitated by Q-PCR.

The addition of retinoic acid at various doses, in combination withFGF-10 at 50 ng/ml, induced differential gene expression patterns thatcorrelate with specific anterior-posterior positional patterns. Thehighest dose of RA (1 μM) preferentially induced expression of anteriorendoderm marker (HOXA3) and also produced the most robust increase inPDX1 (FIGS. 38A-38B). The middle dose of RA (0.2 μM) induced midgutendoderm markers (CDX1, HOXC6) (see FIGS. 38C and 41E), while the lowestdose of RA (0.04 μM) preferentially induced a marker of hindgut endoderm(HOXA13) (see FIG. 38D). The RA dose had essentially no effect on therelative expression of either neural (SOX1) or neuronal (NFM) markers(see FIGS. 38F-38G). This example highlights the use of RA as amorphogen in vitro and in particular as a morphogen of endodermderivatives of differentiating hESCs.

Example 15 Use of B27 Supplement Enhances Expression of PDX1

PDX1 expression in definitive endoderm can be influenced by the use of anumber of factors and cell growth/differentiation conditions. In thefollowing experiment, we show that the use of B27 supplement enhancesthe expression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hES cells grown on mouseembryonic fibroblast feeders with high dose activin A (100-200 ng/ml in0.5-2% FBS/DMEM/F12) for 4 days. The no activin A control received0.5-2% FBS/DMEM/F12 with no added activin A. At four days, culturesreceived either no activin A in 2% FBS (none), and in 2% serumreplacement (SR), or 50 ng/ml activin A together with 2 μM RA and 50ng/ml FGF-10 in 2% FBS/DMEM/F12 (none, +FBS, +B27) and similarly in 2%Serum replacement (SR). B27 supplement, (Gibco/BRL), was added as a 1/50dilution directly into 2% FBS/DMEM/F12 (+B27). Duplicate cell sampleswhere taken for each point, and total RNA was isolated and subjected toQ-PCR as previously described.

FIGS. 39A-39E shows that serum-free supplement B27 provided anadditional benefit for induction of PDX1 gene expression withoutinducing an increase in the expression of markers genes not specific forforegut endoderm as compared to such marker gene expression in cellsgrown without serum.

Example 16 Use of Activin B to Enhance Induction of PDX1

This Example shows that the use of activin B enhances thedifferentiation of PDX1-negative cells to PDX1-positive cells in invitro cell culture.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (50 ng/ml) in low serum/RPMIfor 6 days. The FBS dose was 0% on day one, 0.2% on day two and 2% ondays 3-6. The negative control for definitive endoderm production (NF)received 2% FBS/RPMI with no added activin A. In order to induce PDX1expression, each of the cultures received retinoic acid at 2 μM in 2%FBS/RPMI on day 6. The cultures treated with activin A on days onethrough five were provided with different dosing combinations of activinA and activin B or remained in activin A alone at 50 ng/ml. The noactivin A control culture (NF) was provided neither activin A noractivin B. This RA/activin treatment was carried out for 3 days at whichtime PDX1 gene expression was measured by Q-PCR from duplicate cellsamples.

FIG. 40A shows that the addition of activin B at doses ranging from10-50 ng/ml (a10, a25 and a50) in the presence of 25 ng/ml (A25) or 50ng/ml (A50) of activin A increased the PDX1 expression at least 2-foldover the culture that received only activin A at 50 ng/ml. The increasein PDX1 as a result of activin B addition was without increase in HNF6expression (see FIG. 40B), which is a marker for liver as well aspancreas at this time in development. This result suggests that theproportion of cells differentiating to pancreas had been increasedrelative to liver.

Example 17 Use of Serum Dose to Enhance Induction of PDX1

The expression of PDX1 in definitive endoderm cells is influenced by theamount of serum present in the cell culture throughout thedifferentiation process. The following experiment shows that the levelof serum in a culture during the differentiation of hESCs toPDX1-negative definitive endoderm has an effect on the expression ofPDX1 during further differentiation of these cells to PDX1-positiveendoderm.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.1% on day one, 0.5% on day twoand either 0.5%, 2% or 10% on days 3-5. The no activin A control (NF)received the same daily FBS/RPMI dosing, but with no added activin A.PDX1 expression was induced beginning at day 6 by the addition of RA.During days 6-7, cultures received retinoic acid at 2 μM in 0.5%FBS/RPMI, 1 μM on day 8 and 0.2 μM on day 9-11. The activin A waslowered to 50 ng/ml during retinoic acid treatment and was left absentfrom the no activin A control (NF).

FIG. 41A shows that the FBS dosing during the 3 day period of definitiveendoderm induction (days 3, 4 and 5) had a lasting ability to change theinduction of PDX1 gene expression during the retinoic acid treatment.This was without significant alteration in the expression pattern ofZIC1 (FIG. 41B) or SOX7 (FIG. 41C) gene expression.

Example 18 Use of Conditioned Medium to Enhance Induction of PDX1

Other factors and growth conditions which influence the expression ofPDX1 in definitive endoderm cells were also studied. The followingexperiment shows the effect of conditioned media on the differentiationof PDX1-negative definitive endoderm cells to PDX1-positive endodermcells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.2% on day one, 0.5% on day twoand 2% on days 3-5.

The definitive endoderm cultures generated by 5 days of activin Atreatment were then induced to differentiate to PDX1 expressing endodermby the addition of RA in 2% FBS/RPMI containing activin A at 25 ng/mlfor four days. The RA was 2 μM for the first two days of addition, 1 μMon the third day and 0.5 μM on the fourth day. This base medium for PDX1induction was provided fresh (2A25R) or after conditioning for 24 hoursby one of four different cell populations. Conditioned media (CM) weregenerated from either mouse embryonic fibroblasts (MEFCM) or from hESCsthat were first differentiated for 5 days by one of three conditions; i)3% FBS/RPMI (CM2), or ii) activin A (CM3) or iii) bone morphogenicprotein 4 (BMP4) (CM4). Activin A or BMP4 factors were provided at 100ng/ml under the same FBS dosing regimen described above (0.2%, 0.5%,2%). These three different differentiation paradigms yield three verydifferent populations of human cells by which the PDX1 induction mediacan be conditioned. The 3% FBS without added growth factor (NF) yields aheterogeneous population composed in large part of extraembryonicendoderm, ectoderm and mesoderm cells. The activin A treated culture(A100) yields a large proportion of definitive endoderm and the BMP4treated culture (B100) yields primarily trophectoderm and someextraembryonic endoderm.

FIG. 42A shows that PDX1 was induced equivalently in fresh andconditioned media over the first two days of RA treatment. However, bythe third day PDX1 expression had started to decrease in fresh media andMEF conditioned media treatments. The differentiated hESCs producedconditioned media that resulted in maintenance or further increases inthe PDX1 gene expression at levels 3 to 4-fold greater than fresh media.The effect of maintaining high PDX1 expression in hESC-conditioned mediawas further amplified on day four of RA treatment achieving levels 6 to7-fold higher than in fresh media. FIG. 42B shows that the conditionedmedia treatments resulted in much lower levels of CDX1 gene expression,a gene not expressed in the region of PDX1 expressing endoderm. Thisindicates that the overall purity of PDX1-expressing endoderm was muchenhanced by treating definitive endoderm with conditioned mediagenerated from differentiated hESC cultures.

FIG. 43 shows that PDX1 gene expression exhibited a positive doseresponse to the amount of conditioned media applied to the definitiveendoderm cells. Total volume of media added to each plate was 5 ml andthe indicated volume (see FIG. 43) of conditioned media was diluted intofresh media (A25R). It is of note that just 1 ml of conditioned mediaadded into 4 ml of fresh media was still able to induce and maintainhigher PDX1 expression levels than 5 ml of fresh media alone. Thissuggests that the beneficial effect of conditioned media for inductionof PDX1 expressing endoderm is dependent on the release of somesubstance or substances from the cells into the conditioned media andthat this substance(s) dose dependently enhances production ofPDX1-expressing endoderm.

Example 19 Validation of Antibodies Which Bind to PDX1

Antibodies that bind to PDX1 are useful tools for monitoring theinduction of PDX1 expression in a cell population. This Example showsthat rabbit polyclonal and IgY antibodies to PDX1 can be used to detectthe presence of this protein.

In a first experiment, IgY anti-PDX1 (IgY α-PDX1) antibody binding toPDX1 in cell lysates was validated by Western blot analysis. In thisanalysis, the binding of IgY α-PDX1 antibody to 50 μg of total celllysate from MDX12 human fibroblasts or MDX12 cells transfected 24 hrspreviously with a PDX1 expression vector was compared. The cell lysatesseparated by SDS-PAGE, transferred to a membrane by electroblotting, andthen probed with the IgY α-PDX1 primary antiserum followed by alkalinephosphatase conjugated rabbit anti-IgY (Rb α-IgY) secondary antibodies.Different dilutions of primary and secondary antibodies were applied toseparate strips of the membrane in the following combinations: A (500×dilution of primary, 10,000× dilution of secondary), B (2,000×,10,000×), C (500×, 40,000×), D (2,000×, 40,000), E (8,000×, 40,000×).

Binding was detected in cells transfected with the PDX1 expressionvector (PDX1-positive) at all of the tested antibody combinations.Binding was only observed in untransfected (PDX1-negative) fibroblastswhen using the highest concentrations of both primary and secondaryantibody together (combination A). Such non-specific binding wascharacterized by the detection of an additional band at a molecularweight slightly higher than PDX1 in both the transfected anduntransfected fibroblasts.

In a second experiment, the binding of polyclonal rabbit anti-PDX1 (Rbα-PDX1) antibody to PDX1 was tested by immunocytochemistry. To produce aPDX1 expressing cell for such experiments, MS1-V cells (ATCC # CRL-2460)were transiently transfected with an expression vector of PDX1-EGFP(constructed using pEGFP-N1, Clontech). Transfected cells were thenlabeled with Rb α-PDX1 and α-EGFP antisera. Transfected cells werevisualized by both EGFP fluorescence as well as α-EGFPimmunocytochemistry through the use of a Cy5 conjugated secondaryantibody. PDX1 immunofluorescence was visualized through the use of anα-Rb Cy3-conjugated secondary antibody.

Binding of the Rb α-PDX1 and the α-EGPF antibodies co-localized with GPFexpression.

Example 20 Immunocytochemistry of Human Pancreatic Tissue

This Example shows that antibodies having specificity for PDX1 can beused to identify human PDX1-positive cells by immunocytochemistry.

In a first experiment, paraffin embedded sections of human pancreas werestained for insulin with guinea pig anti-insulin (Gp α-Ins) primaryantibody at a 1/200 dilution followed by dog anti-guinea pig (D α-Gp)secondary antibody conjugated to Cy2 at a 1/100 dilution. In a secondexperiment, the same paraffin embedded sections of human pancreas werestained for PDX1 with IgY α-PDX1 primary antibody at a 1/4000 dilutionfollowed Rb α-IgY secondary antibody conjugated to AF555 at a 1/300dilution. The images collected from the first and second experimentswhere then merged. In a third experiment, cells that were stained withIgY α-PDX1 antibodies were also stained with DAPI.

Analysis of the human pancreatic sections revealed the presence ofstrong staining of islets of Langerhans. Although the strongest PDX1signal appeared in islets (insulin-positive), weak staining was alsoseen in acinar tissue (insulin-negative). DAPI and PDX1 co-stainingshows that PDX1 was mostly but not exclusively localized to the nucleus.

Example 21 Immunoprecipitation of PDX1 from Retinoic Acid Treated Cells

To further confirm PDX1 expression in definitive endoderm cells thathave been differentiated in the presence of RA and the lack of PDX1 indefinitive endoderm cells that have not been differentiated with RA, arabbit anti-PDX1 (Rb α-PDX1) antibody was used to immunoprecipitate PDX1from both RA differentiated and undifferentiated definitive endodermcells Immunoprecipitated RA was detected by Western blot analysis usingIgY α-PDX1 antibody.

To obtain undifferentiated and differentiated definitive endoderm celllysates for immunoprecipitation, hESCs were treated for 5 days withactivin A at 100 ng/ml in low serum (definitive endoderm) followed bytreatment with activin A at 50 ng/ml and 2 μM all-trans RA for two days,1 μM for one day and 0.2 μM for one day (PDX1-positive foregutendoderm). As a positive control cell lysates were also prepared fromMS1-V cells (ATCC # CRL-2460) transfected with a PDX1 expression vector.PDX1 was immunoprecipitated by adding Rb α-PDX1 and rabbit-specificsecondary antibodies to each lysate. The precipitate was harvested bycentrifugation Immunoprecipitates were dissolved in SDS-containingbuffer then loaded onto a polyacrylamide gel. After separation, theproteins were transferred to a membrane by electroblotting, and thenprobed with the IgY α-PDX1 primary antibody followed by labeled Rb α-IgYsecondary antibodies.

Immunoprecipitates collected from the MS1-V positive control cells aswell as those from day 8 (lane d8, three days after the start of RAtreatment) and day 9 (lane d9, four days after the start of RA) cellswere positive for PDX1 protein (FIG. 44). Precipitates obtained fromundifferentiated definitive endoderm cells (that is, day 5 cells treatedwith activin A—designated (A) in FIG. 44) and undifferentiated hESCs(that is, untreated day 5 cells—designated as (NF) in FIG. 44) werenegative for PDX1.

Example 22 Generation of PDX1 Promoter-EGFP Transgenic hESC Lines

In order to use the PDX1 marker for cell isolation, we geneticallytagged PDX1-positive foregut endoderm cells with an expressible reportergene. This Example describes the construction of a vector comprising areporter cassette which comprises a reporter gene under the control ofthe PDX1 regulatory region. This Example also describes the preparationof a cell, such as a human embryonic stem cell, transfected with thisvector as well as a cell having this reporter cassette integrated intoits genome.

PDX1-expressing definitive endoderm cell lines genetically tagged with areporter gene were constructed by placing a GFP reporter gene under thecontrol of the regulatory region (promoter) of the PDX1 gene. First, aplasmid construct in which EGFP expression is driven by the human PDX1gene promoter was generated by replacing the CMV promoter of vectorpEGFP-N1 (Clontech) with the human PDX1 control region (GenbankAccession No. AF192496, the disclosure of which is incorporated hereinby reference in its entirety), which comprises a nucleotide sequenceranging from about 4.4 kilobase pairs (kb) upstream to about 85 basepairs (bp) downstream of the PDX1 transcription start site. This regioncontains the characterized regulatory elements of the PDX1 gene, and itis sufficient to confer the normal PDX1 expression pattern in transgenicmice. In the resulting vector, expression of EFGP is driven by the PDX1promoter. In some experiments, this vector can be transfected intohESCs.

The PDX1 promoter/EGFP cassette was excised from the above vector, andthen subcloned into a selection vector containing the neomycinphosphotransferase gene under control of the phosphoglycerate kinase-1promoter. The selection cassette was flanked by flp recombinaserecognition sites to allow removal of the cassette. This selectionvector was linearized, and then introduced into hESCs using standardlipofection methods. Following 10-14 days of selection in G418,undifferentiated transgenic hESC clones were isolated and expanded.

Example 23 Isolation of PDX1-Positive Foregut Endoderm

The following Example demonstrates that hESCs comprising the PDX1promoter/EGFP cassette can be differentiated into PDX1-positive endodermcells and then subsequently isolated by fluorescence-activated cellsorting (FACS).

PDX1 promoter/EGFP transgenic hESCs were differentiated for 5 days inactivin A-containing media followed by two days in media comprisingactivin A and RA. The differentiated cells were then harvested bytrypsin digestion and sorted on a Becton Dickinson FACS Diva directlyinto RNA lysis buffer or PBS. A sample of single live cells was takenwithout gating for EGFP (Live) and single live cells were gated intoEGFP positive (GFP) and GFP negative (Neg) populations. In oneexperiment, the EGFP positive fraction was separated into two equallysized populations according to fluorescence intensity (Hi and Lo).

Following sorting, cell populations were analyzed by both Q-PCR andimmunocytochemistry. For Q-PCR analysis, RNA was prepared using QiagenRNeasy columns and then converted to cDNA. Q-PCR was conducted asdescribed previously. For immunocytochemistry analysis, cells weresorted into PBS, fixed for 10 minutes in 4% paraformaldehyde, andadhered to glass slides using a Cytospin centrifuge. Primary antibodiesto Cytokeratin19 (KRT19) were from Chemicon; to Hepatocyte nuclearfactor 3 beta (HNF3β) from Santa Cruz; to Glucose Transporter 2 (GLUT2)from R&D systems. Appropriate secondary antibodies conjugated to FITC(green) or Rhodamine (Red) were used to detect binding of the primaryantibodies.

A typical FACS sort of differentiated cells is shown in FIG. 45. Thepercent isolated PDX1-positive cells in this example was approximately7%, which varied depending on the differentiation efficiency from about1% to about 20%.

Sorted cells were further subjected to Q-PCR analysis. Differentiatedcells showed a correlation of EGFP fluorescence with endogenous PDX1gene expression. Compared to non-fluorescing cells, the EGFP positivecells showed a greater than 20-fold increase in PDX1 expression levels(FIG. 46). The separation of high and low EGFP intensity cells indicatedthat EGFP expression level correlated with PDX1 expression level (FIG.47). In addition to PDX1 marker analysis, sorted cells were subjected toQ-PCR analysis of several genes that are expressed in pancreaticendoderm. Products of each of these marker genes (NKX2.2, GLUT2, KRT19,HNF4α and HNF3β) were all enriched in the EGFP positive fraction (FIGS.48A-48E). In contrast, the neural markers ZIC1 and GFAP were notenriched in sorted EGFP expressing cells (FIGS. 49A and 49B).

By immunocytochemistry, virtually all the isolated PDX1-positive cellswere seen to express KRT19 and GLUT2. This result is expected for cellsof the pancreatic endoderm lineage. Many of these cells were also HNF3βpositive by antibody staining.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

REFERENCES

Numerous literature and patent references have been cited in the presentpatent application. Each and every reference that is cited in thispatent application is incorporated by reference herein in its entirety.

For some references, the complete citation is in the body of the text.For other references the citation in the body of the text is by authorand year, the complete citation being as follows:

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1-20. (canceled)
 21. An in vitro cell culture method for producing humananterior foregut endoderm cells, comprising, contacting a population ofhuman pluripotent stem cells with an effective amount of a retinoid toproduce human anterior foregut endoderm cells.
 22. The in vitro cellculture method of claim 21, wherein the retinoid is retinoic acid (RA).23. The in vitro cell culture method of claim 22, wherein the RA isprovided to the population of human pluripotent stem cells at aconcentration of about 1 μM.
 24. The in vitro cell culture method ofclaim 21, wherein the human anterior foregut endoderm cells expressHOXA3.
 25. An in vitro cell culture method for producing human midgutendoderm cells, comprising, contacting a population of human pluripotentstem cells with an effective amount of a retinoid to produce humanmidgut endoderm cells.
 26. The in vitro cell culture method of claim 25,wherein the retinoid is retinoic acid (RA).
 27. The in vitro cellculture method of claim 26, wherein the RA is provided to the populationof human pluripotent stem cells at a concentration of about 0.2 μM. 28.An in vitro cell culture method for producing human hindgut endodermcells, comprising, contacting a population of human pluripotent stemcells with an effective amount of a retinoid to produce human hindgutendoderm cells.
 29. The in vitro cell culture method of claim 28,wherein the retinoid is retinoic acid (RA).
 30. The in vitro cellculture method of claim 29, wherein the RA is provided to the populationof human pluripotent stem cells at a concentration of at least 0.04 μM.31. An in vitro method for producing anterior foregut endoderm cells,midgut endoderm cells and/or hindgut endoderm cells, comprisingcontacting a human pluripotent stem cell population with a retinoid inan amount sufficient to produce the human anterior foregut endodermcells, midgut endoderm cells and/or human hindgut endoderm cells. 32.The in vitro method of claim 31, wherein the retinoid is retinoic acid(RA).
 33. The in vitro method of claim 32, wherein the RA is provided tothe human pluripotent stem cell population at a concentration from about0.04 μM to about 1 μM.
 34. The in vitro method of claim 32, wherein theRA is provided to the human pluripotent stem cell population atconcentration of about 0.04 μM and human hindgut endoderm cells areproduced.
 35. The in vitro method of claim 32, wherein the RA isprovided to the human pluripotent stem cell population at aconcentration of about 0.2 μM and human midgut endoderm cells areproduced.
 36. The in vitro method of claim 32, wherein the RA isprovided to the human pluripotent stem cell population at aconcentration of about 1 μM and human anterior foregut endoderm cellsare produced.
 37. The in vitro method of claim 34, wherein the humanhindgut endoderm cells express HOXA13.
 38. The in vitro method of claim35, wherein the human midgut endoderm cells express HOXC6.
 39. The invitro method of claim 35, wherein the human midgut endoderm cellsexpress CDX1.
 40. The in vitro method of claim 36, wherein the humananterior foregut endoderm cells express HOXA3.